O2-arylated or O2-glycosylated 1-substituted diazen-1-ium-1,2-diolates and O2-substituted 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolates

ABSTRACT

Diazeniumdiolates, wherein the N 1  position is substituted by an inorganic or organic moiety and the O 2 -oxygen is bound to a substituted or unsubstituted aromatic group, are provided. Also provided are O 2 -glycosylated 1-substituted diazen-1-ium-1,2-diolates (O 2 -glycosylated diazeniumdiolates) and O 2 -substituted 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates (1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates). The O 2 -aryl diazeniumdiolates are stable with respect to the hydrolytic generation of nitric oxide in neutral to acidic solutions and generate nitric oxide in basic or nucleophilic environments or microenvironments. Also provided are compositions, including pharmaceutical compositions, comprising such compounds and methods of using such compounds.

This application claim benefit to provisional application No. 60/026,816Sep. 27, 1996 which claims benefit of 60/045,917 May 7, 1997 whichclaims benefit of 60/051,696 Jul. 3, 1997.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to O²-aryl 1-substituteddiazen-1-ium-1,2-diolates (O²-aryl diazeniumdiolates) O²-glycosylated1-substituted diazeniumdiolates, and O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates, compositionscomprising such diazeniumdiolates, methods of using suchdiazeniumdiolates, and methods of preparing O²-aryl diazeniumdiolates.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) has been implicated in a wide variety of bioregulatoryprocesses, and compounds, which contain nitric oxide or are capable ofreleasing nitric oxide, have been identified as useful in regulatingthese processes. Many classes of nitric oxide-containing and/or-releasing adducts are known in the art, such as glyceryl trinitrate andnitroprusside (reviewed in U.S. Pat. No. 5,405,919 (Keefer et al.),including limitations of their use in biological applications). Thelimited utility of such compounds has, in part, given rise to thedevelopment of another class of nitric oxide-generating compounds,diazeniumdiolates, which are especially useful biologically.

Diazeniumdiolates include compounds containing an N₂O₂ ⁻ functionalgroup and are structurally and functionally distinct from nitrosamines(see, e.g., Reilly, U.S. Pat. No. 3,153,094). The knowndiazeniumdiolates are disclosed in recently issued patents. U.S. Pat.Nos. 5,039,705 (Keefer et al.) and 5,208,233 (Keefer et al.) disclosesecondary amine-nitric oxide adducts and salts thereof. U.S. Pat. Nos.5,155,137 (Keefer et al.) and 5,250,550 (Keefer et al.) disclosecomplexes of nitric oxide and polyamines. U.S. Pat. No. 5,389,675(Christodoulou et al.) discloses mixed ligand metal complexes of nitricoxide-nucleophile adducts and U.S. Pat. Nos. 5,525,357 (Keefer et al.)and 5,405,919 (Keefer et al.) disclose polymer-bound nitricoxide/nucleophile adduct compositions. U.S. Pat. Nos. 4,954,526 (Keeferet al.; the '526 patent) and 5,212,204 (Keefer et al.) disclose the useof ionic diazeniumdiolates as cardiovascular agents. In addition, the'526 patent discloses O²-substituted and metal-bound diazeniumdiolates.Keefer et al., U.S. Pat. No. 5,366,997 ('997), disclosesdiazeniumdiolates having the formula:

in which the O²-oxygen of the N₂O₂ ⁻ group is bonded to the functionalgroup R³. When the R³ group is cleaved from the O²-oxygen, NO can bereleased spontaneously.

Although Keefer et al. ('997) discloses that (i) R¹ and R², togetherwith the nitrogen atom to which they are bonded, can form apyrrolidinyl, piperazino or other heterocyclic group, (ii) R³ is a C₁₋₁₂straight-chain or C₃₋₁₂ branched-chain alkyl, optionally olefinic and/orsubstituted with hydroxy, halo, acyloxy or alkoxy, a C₁₋₁₂unsubstituted/substituted acyl, sulfonyl, carboxamido, sulfinyl,sulfenyl, a carbonate derivative or a carbamate derivative, and (iii)the pyrrolidinyl group can have the structure:

wherein w=4, and R⁴=hydrogen, a C₁₋₈ straight or branched chain alkyl, aC₃₋₆ cycloalkyl, or a substituted or an unsubstituted aryl, Keefer etal. ('997) does not disclose that R³ is an aryl or a substituted aryl orthat the pyrrolidino group can be substituted with a substituted orunsubstituted carboxyl group (see, also, Example 1 of U.S. Pat. No.5,632,981) at position 2. Similarly, Keefer et al. ('997) does notdisclose O²-glycosylation of diazeniumdiolates.

Heretofore it was not known that O²-aryl substitutions of thediazeniumdiolates was possible. Further, chemical studies of previouslydisclosed diazeniumdiolates led to the conclusion that they aregenerally at least as stable at high pH as they are at low pH, and that,unlike certain other classes of “nitrovasodilator” drugs, their rates ofNO release are not affected by the presence of nucleophilic thiols.

Thus, there remains a need for such classes of diazeniumdiolates, whichoffer advantages over other currently available diazeniumdiolates. Inthis regard, the O²-aryl substituted diazeniumdiolates are advantageousin that they can release NO spontaneously under alkaline conditions orafter nucleophilic attack. O²-Aryl substituted diazeniumdiolates alsocan release NO spontaneously after a combination of oxidative orelectophilic activation and nucleophilic attack.

It is, therefore, a principal object of the present invention to providea nitric oxide/nucleophile adduct in which the O²-oxygen of the N₂O₂ ⁻group is derivatized with an aryl or substituted aryl group to protectthe diazeniumdiolate against the spontaneous release of NO. It isanother object of the invention to provide a novel class ofdiazeniumdiolates, which resists releasing nitric oxide in neutral oracidic solutions, but releases NO on nucleophilic attack or onincreasing the pH. It is still another object of the present inventionto provide O²-glycosylated 1-substituted diazen-1-ium-1,2-diolates andO²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates. It is afurther object of the present invention to provide compositionscomprising such compounds, including compositions comprising a nitricoxide/nucleophile adduct comprising a novel targeting moiety. It is arelated object to provide O²-aryl substituted diazeniumdiolates, whichare amenable to biological tissue-targeting strategies, which offergreater flexibility and specificity for targeting NO release. It is astill further object of the present invention to provide methods ofusing such compounds. These and other objects of the present invention,as well as additional inventive features, will be apparent from thedescription of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an O²-aryl substituted diazeniumdiolate(i.e., O²-aryl diazeniumdiolate) illustrated by the formula:

wherein X is an inorganic or organic moiety and Q is an aryl moiety. Inthis novel class of compounds an atom of the aryl ring moiety Q isbonded to the O²-oxygen of the N₂O₂ ⁻ functional group. Thediazeniumdiolates of Formula (I) are stable with respect to thehydrolytic generation of nitric oxide in neutral to acidic solutions.Surprisingly, these novel compounds, or the resultant product of thesecompounds after oxidative or electrophilic activation, have provencapable of generating nitric oxide in basic or nucleophilicenvironments, in which the aryl moiety is separated from the remainderof the diazeniumdiolate.

The present invention also provides O²-glycosylated 1-substituteddiazen-1-ium-1,2-diolates and O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates, both ofwhich can be represented by the formula:

in which X and R are organic and/or inorganic moieties as definedherein, although for O²-glycosylated diazeniumdiolates, R must be asaccharide.

Further with respect to the O²-glycosylated 1-substituteddiazen-1-ium-1,2-diolates, the moiety X can be any organic or inorganicgroup. Preferably, X contains atoms other than carbon and hydrogen, andis linked to the nitrogen of the diazeniumdiolate through an atom otherthan carbon. Most preferably, X is an amino group, and is linked to thenitrogen of the diazeniumdiolate through a nitrogen atom.

With respect to the O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates, X ofFormula Ia can be

such that the [1-(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates can bestructurally represented by the formula:

wherein R²² is hydrogen, hydroxyl, OM, wherein M is a cation, halo, orX¹R²³R²⁴, wherein X¹ is oxygen, nitrogen or sulfur and R²³ and R²⁴ areindependently a substituted or unsubstituted C₁₋₂₄ alkyl, a substitutedor unsubstituted C₃₋₂₄ cycloalkyl, a substituted or unsubstituted C₂₋₂₄olefinic, a substituted or unsubstituted aryl (such as acridine,anthracene, benzene, benzofuran, benzothiophene, benzoxazole,benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, furan,imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole,isoquinoline, naphthalene, oxazole, phenanthrene, phenanthridine,phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine,porphin, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrocoline, pyrrole, quinolizinium ion, quinoline,quinoxaline, quinazoline, sydnone, tetrazole, thiazole, thiophene,thyroxine, triazine, and triazole), or a heterocyclic group, such asglycosyl, and the like, and when X¹ is O or S, there is no R²⁴ group.Alternatively, when X¹ is nitrogen, R²³ and R²⁴, together with X¹, forma heterocyclic ring, such as a heterocyclic ring selected from the groupconsisting of:

in which A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸, R⁹, R²⁵,and R²⁶ are hydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chainalkyl, a C₃₋₈ cycloalkyl, or an aryl. The aforementioned R²³ and R²⁴groups can be unsubstituted or substituted as appropriate. For example,the R²³ and R²⁴ groups can be substituted as appropriate with acyloxy,acylthio, hydroxyl, amino, carboxyl, mercapto, halo, amido, sulfonyl,sulfoxy, sulfenyl, phosphono, phosphate, and the like.

Further with respect to the O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates, the moietyR of Formula Ia can be any organic or inorganic moiety, which iscovalently bound to the terminal oxygen of the diazeniumdiolate as shownbut which is other than hydrogen and is a substituted or unsubstitutedC₁₋₁₂ straight chain or C₃₋₁₂ branched chain alkyl, a substituted orunsubstituted C₂₋₁₂ straight chain or C₃₋₁₂ branched chain olefinic, asubstituted or unsubstituted C₁₋₁₂ acyl, sulfonyl, carboxamido, aglycosyl group, an aryl group, or a group of the formula —(CH₂)_(n)—ON═N(O)NR²⁸R²⁹, wherein n is an integer of 2-8, and R²⁸ and R²⁹ areindependently a C₁₋₁₂ straight chain alkyl, a C₃₋₁₂ branched chainalkyl, a C₂₋₁₂ straight chain or C₃₋₁₂ branched chain olefinic, or R²⁸and R²⁹, together with the nitrogen atom to which they are bonded, forma heterocyclic group, preferably a pyrrolidino, piperidino, piperazinoor morpholino group. The aforementioned R groups can be unsubstituted orsubstituted. Preferred substitutions include those made with hydroxy,halo, acyloxy, alkoxy, acylthio, or benzyl.

In another aspect, the present invention comprises a composition,including a pharmaceutical composition, comprising a present inventivediazeniumdiolate. The pharmaceutical composition preferably additionallycomprises a pharmaceutically acceptable carrier.

In yet another aspect, the present invention provides methods of using acompound in accordance with the present invention.

In still another aspect, the present invention provides a method ofmaking O²-aryl diazeniumdiolates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of Trp37 fluorescence (RFU) versus time (min), whichdepicts zinc ejection from HIV-1 nucleocapsid p7 protein by O²-aryldiazeniumdiolates. In the graph, ◯ represents the negative control,i.e., no drug, □ represents the positive control, i.e., 624151 (See Riceet al., Antimicrob. Agents Chemother. 41: 419426 (1997)), ▪ representsthe compound of Example 1 (LK1), ♦ represents the compound of Example 8(LK2), ▴ represents the compound of Example 5 (LK3),  represents thecompound of Example 10 (LK4), and X represents the compound of Example11 (LKS).

FIG. 2 is a graph of relative NO release rate versus time (min), whichdepicts the catalysis of NO release from DNP-PYRRO/NO by glutathioneS-transferase (GST).

DETAILED DESCRIPTION OF THE INVENTION

O²-arylated diazeniumdiolates

The present invention provides an O²-aryl 1-substituted diazeniumdiolate(i.e., O²-aryl 1-substituted diazen-1-ium-1,2-diolate) having theformula:

wherein X is an organic or inorganic moiety and Q is an aryl group.

In accordance with the invention, the O²-oxygen of the N₂O₂ ⁻ group isbonded directly to an atom of the ring of the aryl group. Stated anotherway, there are no spacer atoms (e.g., methylene) that separate theO²-oxygen from the aryl ring. If the aryl group comprises a bicyclic orpolycyclic moiety and all rings of the aryl group are not aromatic, thenthe linkage between the O²-oxygen and the aryl group is through an atomthat is part of an aromatic ring. Further, the O²-oxygen can be linkedto any aromatic, ring atom of the aryl group that is capable of bondingto the O²-oxygen of the N₂O₂ ⁻ group. Atoms of the aromatic ring thatare capable of bonding with the O²-oxygen of the N₂O₂ ⁻ group aretypically carbon and nitrogen, although there can be other linkages aswell.

While not wishing to be bound to any particular theory, it is presentlybelieved that the bonding of the O²-oxygen with the atom of the arylring is accomplished by bonding to an activated atom of the ring.Activation can be accomplished through any suitable mechanism. In thisregard, a preferred mechanism of activating an aryl ring is by reactingthe diazeniumdiolate through an atom of the aryl ring possessing apartial positive charge or, more specifically, by displacing an aminosubstituent of the ring structure.

In the first preferred reaction mechanism, the aryl ring is substitutedby a suitable electron-withdrawing group(s), which can be part of thering, as in Example 12, and a “leaving group” prior to reaction with thediazeniumdiolate. It will be appreciated by those skilled in the artthat the electron-withdrawing group and the leaving group can, in someinstances, be the same moiety. The leaving group is displaced by thediazeniumdiolate to form the O²-aryl diazeniumdiolate of the presentinvention. Suitable leaving groups include, but are not limited to, F,Cl, Br, I, NO₂, OSO₂R, and OSO₃R, wherein R is an organic moiety, ametal center, or the like, the composition of which is well understoodby those skilled in the art. By way of illustration and not inlimitation, suitable R groups include H, alkyl, alkenyl, or aryl. Thisreaction mechanism is based on the well known S_(N)Ar mechanism; forexample, see Nucleophilic Aromatic Displacement: The Influence of theNitro Group, Francois Terrier, VCH Publishing, Inc., New York, N.Y.pages 1-11 (1991). Preferably, these S_(N)Ar reactions are carried outin electron-deficient aromatic rings comprising at least oneelectron-withdrawing group.

In the second preferred reaction mechanism, an aryl reactant issubstituted by a suitable amino group, which allows directderivatization (e.g., after diazotization of the amino group) of thering atom of the aryl group that is bound to the displaced amino group.There is no requirement for the atom of the aryl ring linked to theO²-oxygen to be activated after it has been incorporated into thepresent inventive compound. However, if this atom is activated afterbeing incorporated into the present inventive compound, then thediazeniumdiolate moiety to which it is bound may be displaced throughfurther nucleophilic displacement (e.g., in a suitably strong base).Alternatively, an oxidative or electrophilic activation event can alterthe present inventive compound so that the aryl ring atom linked to theO²-oxygen becomes activated, thereby rendering the compound subject tofurther nucleophilic displacement, as observed above.

Advantageously, the compounds of the present invention have new anduseful properties, which are not possessed by other nitricoxide/nucleophile adducts previously known in the art. In general, thecompounds of the present invention are stable at neutral or acidic pH(i.e., at neutral or acidic pH, the compounds indicated by Formula I donot generate NO). Another advantageous property of the compounds of thepresent invention is that the O²-aryl linkage is often susceptible tocleavage by nucleophiles, including hydroxide ions. When the typicalO²-aryl diazeniumdiolate or the oxidatively or electrophilicallyactivated O²-aryl diazeniumdiolate of the present invention is placedinto a basic or nucleophilic environment, the aryl linkage to theO²-oxygen can be broken. The resulting diazeniumdiolate ionspontaneously degrades via a predictable, first order mechanism, givingrise to NO. The resulting aryl group is substituted with a nucleophileprovided by the environment. If the nucleophile provided by theenvironment is part of an enzyme, that enzyme can be inactivated. Thesusceptibility to nucleophilic attack of the O²-aryl diazeniumdiolatesalso makes them particularly amenable to designing prodrugs fortargeting nitric oxide to nucleophilic tissue components, body sites andmicroenvironments in the body.

The compounds of the present invention are also useful to identify andquantify individual thiols (organic —SH containing compounds) when thethiols are present in mixtures. For example, a sample suspected toconsist of C₄-C₈ straight-chain thiols can be analyzed by dissolving theproduct of Example 1 in tetrahydrofuran or another inert solvent, thenmixing a molar excess of the resulting solution with the sample to beassayed. After the ensuing reaction is complete, an aliquot is subjectedto HPLC analysis using an ultraviolet detection system. Peaks found inthe resulting chromatogram can be identified by comparing theirretention times to those of independently derivatized authenticstandards of the individual C₄-C₈ straight-chain thiols, and quantifiedby transforming peak areas to concentrations via the individual standardcurves.

With respect to the O²-aryl diazeniumdiolates, “aryl group” as usedherein refers to any aromatic group, regardless of whether it is part ofa (homo)cyclic, heterocylic, or polycyclic structure. The standardunderstanding of “aromatic” is used herein (See, e.g., L. G. Wade, Jr.,Organic Chemistry, 2d Edition, Prentice Hall, Englewood Cliffs, N.J.,682-683 (1991)). The aryl group, as used herein, can also have a widevariety of substituent groups. Any suitable aryl substituent can be usedproviding that the substituent does not destroy the aromaticity of thearyl ring.

Turning to the aryl group Q of Formula I, Q is intended to include allaryl groups that are (or can be made) amenable to reaction with theO²-oxygen atom of a diazeniumdiolate. The moiety Q thus includeshomocyclic, heterocyclic, and polycyclic aromatic structures as well asderivatives thereof. Illustrative of the aryl groups Q are acridine,anthracene, benzene, benzofuran, benzothiophene, benzoxazole,benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, furan,imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole,isoquinoline, naphthalene, oxazole, phenanthrene, phenanthridine,phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine,porphin, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrocoline, pyrrole, quinolizinium ion, quinoline,quinoxaline, quinazoline, sydnone, tetrazole, thiazole, thiophene,thyroxine, triazine, and triazole.

In keeping with the invention, each of these aromatic compounds Q can bevariably derivatized with the numerous substituents well known in theart that are capable of being substituted into an aromatic ring so longas the aromaticity of the ring is maintained. For example, thesubstituents of the aryl moiety, Q, can include X[N(O)NO]⁻, wherein X isas defined hereinafter and is the same as X of Formula I, halo, hydroxy,alkylthio, arylthio, alkoxy, aryloxy, amino, mono- or di-substitutedamino, ammonio or substituted ammonio, nitroso, cyano, sulfonato,mercapto, nitro, oxo, C₁-C₂₄ aliphatic, C₃-C₁₂ oleinic, C₃-C₂₄,cycloalkyl, C₃-C₂₄ heterocycloalkyl, benzyl, phenyl, substituted benzyl,substituted phenyl, benzylcarbonyl, phenylcarbonyl, saccharides,substituted benzylcarbonyl, substituted phenylcarbonyl and phosphorusderivatives. Illustrative phosphorus derivatives include phosphate andphosphono moieties. Illustrative phosphate moieties include (OH)₂P(O)O—and substituted (OH)₂P(O)O-moieties, wherein one or more oxygen atomscan be independently replaced by S or NR′, wherein R′ is understood tobe a C₁-C₁₀ containing aliphatic, cycloalkyl, or aryl group.Illustrative C₁-C₂₄ aliphatic substituents comprise C₁-C₂₄ acyl, and

wherein R is hydrogen, substituted or unsubstituted C₁-C₂₃ aliphatic,substituted or unsubstituted C₃-C₂₃ cycloalkyl, substituted orunsubstituted C₃-C₁₂ olefinic, benzyl, phenyl, substituted benzyl orsubstituted phenyl, and said substituted benzyl or substituted phenyl issubstituted with one to five substituents selected from the groupconsisting of nitro, halo, hydroxy, C₁-C₂₄ alkyl, C₁-C₂₄ alkoxy, amino,mono-C₁-C₂₄ alkylamino, di-C₁-C₂₄ alkylamino, cyano, phenyl and phenoxy.Preferred saccharides include ribose, glucose, deoxyribose, dextran,starch, glycogen, lactose, fucose, galactose, fructose, glucosamine,galactosamine, heparin, mannose, maltose, sucrose, sialic acid, andcellulose. Other preferred saccharides are phosphorylated,3,5-cyclophosphorylated, and polyphosphorylated hexoses and pentoses.

Examples of substituted aryl compounds of the present invention that canbe linked to the diazeniumdiolate group comprise dinitrophenol (abenzene), hypoxanthine (a purine), uridine (a pyrimidine), vitamin K₅ (anaphthalene) and ribosyl uridine (a nucleoside).

In another particular embodiment of the present invention, the arylmoiety is identical to or structurally analogous to molecules, orsubstituents thereof, normally found in living organisms. Thesebiologically relevant groups can be selected from nucleotides,nucleosides, and nucleic acids, peptides, including peptide hormones,non-peptide hormones, vitamins and other enzyme cofactors such asporphyrins, and others. Examples of biologically relevant aryl groupsare thyroxine, NAD (or NADH), chlorophyll, hypoxanthine, uridine, andvitamin K₅.

The following reaction schematics illustrate methods of preparing theO²-aryl diazeniumdiolates of the present invention. In theseillustrative reactions, in general, a solution of a diazeniumdiolate(X—[N₂O₂ ⁻]) in 5% aqueous sodium bicarbonate. (which is weakly basic)is cooled to 0° C., preferably under a blanket of inert gas such asnitrogen. A solution containing one equivalent of the activated aromaticreagent in a solvent, such as t-butyl alcohol, dimethyl sulfoxide, orN,N-dimethylformamide, is then added slowly. While not being bound toany particular theory, it is believed that polar non-protic solvents arepreferred. The reaction temperature is raised slightly for less reactivearyl moieties, for example, to ambient temperatures or higher.Generally, a precipitate forms upon addition. The mixture is thenallowed to warm to room temperature gradually and stirred overnight. Theproduct may be extracted with a suitable extraction agent, such asdichloromethane, and washed subsequently with cold dilute hydrochloricacid and then with sodium bicarbonate solution. The organic layer isdried over a suitable drying agent, such as sodium sulfate, filtered,preferably through a layer of anhydrous magnesium sulfate, andevaporated under vacuum to give the crude product. Usually, the productis solid. Recrystallization from ethanol or other suitable solvents is apreferred method of purifying the product. It will be appreciated by oneskilled in the art that these conditions can be modified to suit theparticular application of the artisan. Accordingly, alternative methodsof preparation are also embraced.

Chlorinated quinoline and isoquinoline can be reacted with adiazeniumdiolate such that the Cl substituent is replaced by theO²-oxygen of a diazeniumdiolate, as shown below:

Additionally, quinazoline can be incorporated as shown:

The phthalazines also are incorporated in accordance with the presentinvention, as indicated:

Acridine can be incorporated as indicated:

Cinnoline can also be incorporated as indicated:

Quinoxaline can also be incorporated as indicated:

Oxygen- and sulfur-containing heteroaromatics can also be used as thearomatic reagent for O²-oxygen substitution of the diazeniumdiolate inaccordance with the present invention. For example, oxazole andbenzoxazole can be derivatized at the 2-position as indicated:

Similarly, thiazole and benzothiazole can also be derivatized at the2-position.

A derivatized Vitamin K₇ can also be prepared, as indicated:

The O²-diazeniumdiolated atom of the aryl ring in the right-most(directly above) structure is not activated. Therefore, the right-moststructure is resistant to nucleophilic attack, which would re-generateX—N₂O₂ ⁻, which, in turn, would spontaneously degrade to produce NO.Therefore, the right-most structure must undergo oxidative preactivationprior to nucleophilic attack in order to generate NO. This oxidativepreactivation requirement would also be of advantage in targeting a cellor organ type that is uniquely able to perform the required oxidation,thereby limiting NO exposure to the desired tissue while avoidingexposure at other NO— sensitive portions of the anatomy.

Illustrative of the class of compounds requiring electrophilicpreactivation is the compound indicated below:

Triazines can likewise be the aromatic reagent that forms the O²-arylsubstituted diazeniumdiolates of the present invention as shown below.The synthesis of such compounds should enhance the potency of existingtriazine-derived herbicides.

Nucleic acids and the nitrogenous bases they comprise (includingribosylated bases) can also be used as the aromatic reagent to form theO²-aryl substituted diazeniumdiolates of the present invention. This isillustrated in Example 13.

Another interesting O²-arylated diazeniumdiolate is the one shown as theproduct in the reaction below; it can co-generate NO and allopurinol onhydrolysis.

Advantageously, allopurinol is already known to be pharmaceuticallyuseful. Thus, by converting known pharmaceutically useful compoundscontaining a suitable aryl group to the O²-aryl diazeniumdiolates of thepresent invention, the present invention allows existing drugs to beenhanced by the release of NO.

Similarly, a derivative of a biopterin diazeniumdiolate can be preparedfrom a substituted pteridine, as indicated below.

An example of a suitable aryl substitution that utilizes linkage througha heteroatom is shown in the following scheme which can be effected byreaction with BuONO or other suitable nitrosating agents.

A structural analog of Bendazac, a well-known anti-inflammatory agent,can be prepared as indicated:

In accordance with the invention, any of the compounds in the class ofcompounds defined as diazeniumdiolates can be subjected to O²-arylsubstitution. Thus, for the compounds having Formula I, X can be anyorganic or inorganic moiety. Preferably, X contains atoms other thancarbon and hydrogen, and is linked to the nitrogen of the N₂O₂ ⁻ groupthrough an atom other than carbon. Most preferably, X is an amine, andis linked to the nitrogen of the N₂O₂ ⁻ group through a nitrogen atom.Suitable moieties of X also include, but are not limited to, C₁-C₂₄aliphatic, aryl, and nonaromatic cyclic. By “aliphatic” is meant acyclicmoieties containing carbon and hydrogen and optionally containingnitrogen, oxygen, sulfur, phosphorus, and halogens. By “aryl” is meant,as hereinabove, a moiety containing at least one aromatic ring.Preferably, the aryl moiety is a C₃-C₃₀-containing moiety. Bynon-aromatic cyclic is meant a moiety containing at least one ringstructure and no aromatic rings. Preferably, the non-aromatic cyclicmoiety is a C₃-C₃₀-containing moiety.

The moiety X of Formula I can be unsubstituted or substituted withsuitable additional moieties, such as, for example, —[N(NO)O—], halo,hydroxy, alkylthio, alkoxy, aryloxy, amino, mono- or di-substitutedamino, cyano, sulfonato, mercapto, nitro, substituted or unsubstitutedC₁-C₁₂ aliphatic, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted C₃-C₈ heterocycloalkyl, substituted orunsubstituted C₃-C₁₂ olefinic, benzyl, phenyl, substituted benzyl,substituted phenyl, benzylcarbonyl, phenylcarbonyl, saccharides,substituted benzylcarbonyl, substituted phenylcarbonyl and phosphorusderivatives. Illustrative phosphorus derivatives include phosphato andphosphono moieties. Illustrative phosphato moieties include (OH)₂P(O)O—and substituted (OH)₂P(O)O— moieties, wherein one or more oxygen atomscan be independently replaced by S or NR′, wherein R′ is understood tobe a C₁-C₈-containing aliphatic, cycloalkyl, or aryl group. PreferredC₁-C₁₂ aliphatic substituents comprise C₁-C₁₂ acyl, and

wherein R is C₁-C₁₀ substituted or unsubstituted aliphatic, C₃-C₁₁olefinic, C₃-C₈ substituted or unsubstituted cycloalkyl, benzyl, phenyl,substituted benzyl or substituted phenyl, and said substituted benzyl orsubstituted phenyl is substituted with one or two substituents selectedfrom the group consisting of halogen, hydroxy, C₁-C₄ alkyl, C₁-C₄alkoxy, amino, mono-C₁-C₄ alkylamino, di-C₁-C₄ alkylamino, phenyl andphenoxy. Preferred saccharides and polysaccharides include ribose,glucose, deoxyribose, dextran, starch, glycogen, lactose, galactose,fructose, glucosamine, galactosamine, heparin, mannose, maltose,sucrose, sialic acid, and cellulose. Other preferred saccharides arephosphorylated, 3,5-cyclophosphorylated, and polyphosphorylated pentosesand hexoses.

In one embodiment of the invention, X is an inorganic moiety asdescribed in U.S. Pat. No. 5,212,204. Preferred embodiments of FormulaI, in which X is inorganic, are ⁻O₃S— (sulfite) and —O⁻ (oxide).

In another embodiment of the present invention, X is a polyamine asdefined in U.S. Pat. No. 5,155,137. Thus, the polyamine substitutedO²-aryl diazeniumdiolates havethe formula

wherein Q is the same as the Q in Formula I and is defined as above, band d can be the same or different and are zero or one, R¹, R², R³, P⁴,and R⁵ are the same or different and comprise hydrogen, substituted orunsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted C₁-C₁₂straight or branched chain alkyl, substituted or unsubstituted benzyl,substituted or unsubstituted benzoyl, substituted or unsubstitutedC₃-C₁₂ olefinic, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,t-butoxycarbonyl, or 2,2,2-tri-halo-t-butoxycarbonyl. The values of i,j, and k in Formula II can be the same or different and are integersfrom 2 to 12.

In a preferred embodiment of the present invention the O²-aryldiazeniumdiolates are derived from the compounds disclosed in U.S. Pat.Nos. 5,039,705 (Keefer et al.) and 4,954,526 (Keefer et al.) and, thus,have the formula

wherein R⁶ and R⁷ can be the same or different and are chosen from H,C₁-C₁₂ straight chain alkyl, C₁-C₁₂ alkoxy or acyloxy substitutedstraight chain alkyl, C₂-C₁₂ hydroxy or halo substituted straight chainalkyl, C₃-C₁₂ branched chain alkyl, C₃-C₁₂ hydroxy, halo, alkoxy, oracyloxy substituted branched chain alkyl, C₂-C₁₂ straight chain olefinicand C₃-C₁₂ branched chain olefinic, which are unsubstituted or which aresubstituted with hydroxy, alkoxy, acyloxy, halo or benzyl, provided thatboth R⁶ and R⁷ are not H; or R⁶ and R⁷, together with the nitrogen atomto which they are bonded, form a heterocyclic ring selected from thegroup consisting of:

wherein A is N, O, or S, w is 1 to 12, y is 1 or 2, z is 1 to 5, R⁸ ishydrogen, C₁-C₈ straight chain alkyl, C₃-C₈ branched chain alkyl, C₃-C₈cycloalkyl, unsubstituted or substituted aryl, such as phenyl, tolyl orthe like, and R⁹ is hydrogen, C₁-C₆ straight chain alkyl or C₃-C₆branched chain alkyl. Exemplary aza crown groups are 1-aza-12-crown-4,1-aza-15-crown-5, and 1-aza-18-crown-6. Where A is nitrogen, thenitrogen atom, itself, can be substituted, as described, for example, inU.S. application Ser. No. 08/475,732, which is incorporated by referenceherein.

Further examples include the O²-aryl substituted diazeniumdiolatesderived from the compounds disclosed in U.S. Pat. No. 5,250,550,previously incorporated in its entirety by reference, and, thus, havethe formula

wherein D is

and wherein R¹⁰ and R¹¹ are the same or different. The substituents R¹⁰and R¹¹ can be any suitable group, examples of which include hydrogen,C₃-C₈ cycloalkyl, C₁-C₂ straight or branched chain alkyl, benzyl,benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl,and 2,2,2-trihalo-t-butoxycarbonyl. In Formula IV, f is an integer from0 to 12.

Preferred O²-aryl substituted diazeniumdiolates also include those ofExample 14.

An alternative method of preparing O²-arylated diazeniumdiolates ispossible through adaptation of the following literature reaction(Stevens, J.Org. Chem. 29: 311-315 (1964)).

By substituting aryloxy anion ArO⁻ for the methoxide of Stevens'sreaction, it is possible to obtain O²-aryl diazeniumdiolates of variedstructure. Similarly, it is possible to obtain derivatives correspondingto ArS⁻ species.

O²-glycosylated diazeniumdiolates and 1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates

The present invention also provides two other new classes ofdiazeniumdiolates, one class of which contains a hydrolytically labilegroup (R), which, upon cleavage to the free diazeniumdiolate (NO donor)X—NO═NO⁻, releases an innocuous and possibly beneficial saccharide andallows advantage to be taken of saccharide-based receptor-mediatedphenomena. The other class of diazeniumdiolates provides, among others,prodrugs of the salt disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO),which is an ultrafast NO donor of proven effectiveness as anantithrombotic agent and a vasodilator but is inherently extremelydifficult to derivatize, due to its instability (Saavedra et al., J.Med. Chem. 31:4361-4365 (1996); and U.S. Pat. No. 5,632,981 (Saavedra etal.)). The newly discovered ability to generate prodrugs of theultrafast NO donor PROLI/NO allows the PROLI/NO prodrugs to move freelythrough the circulatory system until they reach the desired organ orcell type for metabolic removal of the stabilizing O²-protecting group,thereby providing a rapid release of NO at the specific or preferredsite and obviating the need for administration by infusion at acontrolled rate in the vicinity of the target tissue. Additionally, thecorresponding nitrosamine, N-nitrosoproline, if formed in the biologicalmedium, does not pose a carcinogenic threat, unlike other nitrosamines.

Accordingly, the present invention provides O²-glycosylated1-substituted diazen-1-ium-1,2-diolates (O₂-glycosylateddiazeniumdiolates) and O²-substituted1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolates(1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates), both of which canbe represented by the formula:

In Formula Ia, X and R are organic and/or inorganic moieties as definedherein.

O²-glycosylated diazeniumdiolates

With respect to the O²-glycosylated diazeniumdiolates, any of thecompounds in the class of compounds defined as diazeniumdiolates (seee.g., U.S. Pat. Nos. 5,039,705, 5,208,233, 5,155,137, 5,250,550,5,389,675, 5,525,357, 5,405,919 and related patents and patentapplications) can be subjected to O²-glycosylation, provided that the O²of the diazeniumdiolate is available for glycosylation. The moiety R ofFormula Ia can be any saccharide, which is attached to the O² of thediazeniumdiolate by the 2 position of a pyranose or furanose ring. Thesaccharide can be functionalized. Desirably, the saccharide and itsderivatives are hydrolyzable at physiological pH. The saccharide can bea monosaccharide, disaccharide, such as sucrose or maltose, anoligosaccharide or a polysaccharide. Preferred saccharides andpolysaccharides include, among others, ribose, glucose, deoxyribose,fucose, lactose, galactose, fructose, glucosamine, galactosamine,mannose, maltose, sucrose, and the many saccharide and oligosaccharideunits that serve as recognition sequences in receptor-mediated cellularinteractions. Other preferred saccharides include those that arephosphorylated, 3,5-cyclophosphorylated, and polyphosphorylated pentosesand hexoses.

By way of illustration, the saccharide residue (shown attached to thediazeniumdiolate for illustrative purposes) can be an amino sugar, suchas a glucosamine or a substituted glucosamine having the structure:

wherein R¹² and R¹³ can be the same or different and are a hydrogen, aC₁₋₆ alkyl, an acyl, a phosphate, a sulfate, a peptide or a protein. Thesaccharide residue can be, for example, glucuronic acid or a derivativethereof:

wherein R¹⁴ is X¹R¹⁵R¹⁶, wherein X¹ is N, O or S and, when X¹ is N, R¹⁵and R¹⁶ are independently a hydrogen or a substituted or anunsubstituted C₁₋₂₄ alkyl, C₃₋₂₄ cycloalkyl, C₂₋₂₄ olefinic, aryl. (suchas acridine, anthracene, benzene, benzofuran, benzothiophene,benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll,cinnoline, furan, imidazole, indole, isobenzofuran, isoindole,isoxazole, isothiazole, isoquinoline, naphthalene, oxazole,phenanthrene, phenanthridine, phenothiazine, phenoxazine, phthalimide,phthalazine, phthalocyanine, porphin, pteridine, purine, pyrazine,pyrazole, pyridazine, pyridine, pyrimidine, pyrrocoline, pyrrole,quinolizinium ion, quinoline, quinoxaline, quinazoline, sydnone,tetrazole, thiazole, thiophene, thyroxine, triazine, and triazole), orheterocyclic group, such as glycosyl and the like, and when X¹ is O orS, there is no R¹⁶ group.

Alternatively, when Xl is nitrogen, R¹⁵ and R¹⁶ form a heterocyclic ringselected from the group consisting of:

wherein A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R⁸ ishydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chain alkyl, aC₃₋₈ cycloalkyl, an aryl (such as phenyl, tolyl or the like), orcarboxylato and derivatives thereof as further described herein, and R⁹is hydrogen, a C₁₋₆ straight chain alkyl or a C₃₋₆ branched chain alkyl.

The aforementioned groups can be unsubstituted or substituted asappropriate.

Exemplary aza crown groups (i.e., where A is N) are 1-aza-12-crown-4,1-aza-15-crown-5, and 1-aza-18-crown-6. Where A is nitrogen, thenitrogen atom, itself, can be substituted, as described, for example, inU.S. patent application Ser. No. 08/475,732.

Further with respect to the O²-glycosylated diazeniumdiolates, themoiety attached to the carbonyl group through X¹ can be anything thatdoes not interfere with the cleavage to the diazeniumdiolate anion.

Further with respect to the O²-glycosylated diazeniumdiolates, themoiety attached to the carbonyl group through X¹ can be anything thatdoes not interfere with the cleavage to the diazeniumdiolate anion.

Preferably, the moiety X contains atoms other than carbon and hydrogen,and is linked to the nitrogen of the N₂O₂ ⁻ group through an atom otherthan carbon. Most preferably, X is an amino group, and is linked to thenitrogen of the N₂O₂ ⁻ group through a nitrogen atom. Suitable moietiesof X include, but are not limited to, C₁₋₂₄ aliphatic, aryl andnon-aromatic cyclic groups. By “aliphatic” is meant an acyclic moietycontaining carbon and hydrogen and optionally containing nitrogen,oxygen, sulfur, phosphorus or a halogen. By “aryl” is meant a moietycontaining at least one aromatic ring. Preferably, the aryl moiety is aC₆₋₃₀ moiety. By “non-aromatic cyclic” is meant a moiety containing atleast one ring structure and no aromatic rings. Preferably, thenon-aromatic cyclic moiety is a C₆₋₃₀ moiety. Further, X can beunsubstituted or substituted with suitable additional moieties, such as,for example, —[N(NO)O⁻], a halo, a hydroxy, an alkylthio, an alkoxy, anaryloxy, an amino, a mono- or di-substituted amino, a cyano, asulfonato, a mercapto, a nitro, a substituted or unsubstituted C₁₋₁₂aliphatic, a substituted or unsubstituted C₃₋₈ cycloalkyl, a substitutedor unsubstituted C₃-C₁₂ olefinic, a substituted or unsubstituted C₃₋₈heterocycloalkyl, a benzyl, a phenyl, a substituted benzyl, asubstituted phenyl, a benzylcarbonyl, a phenylcarbonyl, a saccharide, asubstituted benzylcarbonyl, a substituted phenylcarbonyl and aphosphorus derivative. Illustrative phosphorus derivatives includephosphato and phosphono moieties. Illustrative phosphato moietiesinclude (OH)₂P(O)O— and substituted (OH)₂P(O)O— moieties, wherein one ormore oxygen atoms can be independently replaced by S or NR¹⁷, whereinR¹⁷ is understood to be a C₁₋₈ aliphatic, a

wherein R¹⁸ is a C₁₋₁₀ unsubstituted or substituted aliphatic, a C₃₋₈unsubstituted or substituted cycloalkyl, benzyl, phenyl, substitutedbenzyl or substituted phenyl. When the benzyl or phenyl is substituted,preferably it is substituted with one or two substituents selected fromthe group consisting of halogen, hydroxy, a C₁₋₄ alkyl, a C₁₋₄ alkoxy,an amino, a mono-C₁₋₄ alkylamino, a di-C₁₋₄ alkylamino, phenyl andphenoxy.

In one embodiment of the invention, X in Formula Ia is an inorganicmoiety as described in U.S. Pat. No. 5,212,204. Preferred embodiments ofFormula Ia, in which X is inorganic, are ⁻O₃S— (sulfite) and —O⁻(oxide).

In another embodiment of the present invention, X in Formula Ia is apolyamine as defined in U.S. Pat. No. 5,250,550. Thus, the polyamineO²-glycosylated diazeniumdlolates have the formula

wherein Q is the same as the R in Formula Ia and is defined as above, band d can be the same or different and are zero or one, R¹, R², R³, R⁴,and R⁵ are the same or different and are hydrogen, substituted orunsubstituted C₃₋₈ cycloalkyl, substituted or unsubstituted C₁₋₁₂straight or branched chain alkyl, substituted or unsubstituted benzyl,substituted or unsubstituted benzoyl, substituted or unsubstitutedC₃-C₁₂ olefinic, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,t-butoxycarbonyl, or 2,2,2-tri-halo-t-butoxycarbonyl. The values of i,j, and k in Formula II can be the same or different and are integersfrom 2 to 12.

In a preferred embodiment of the present invention, thediazeniumdiolates are derived from the compounds disclosed in U.S. Pat.Nos. 5,039,705 (Keefer et al.) and 4,954,526 (Keefer et al.), and, thus,have the formula

wherein R is the same as the R in Formula Ia and is defined as above,R¹⁹ and R²⁰ are the same or different and are hydrogen, a C₁₋₁₂ straightchain alkyl, a C₃₋₁₂ branched chain alkyl, or a C₂₋₁₂ straight or C₃₋₁₂branched chain olefinic, provided that both R¹⁹ and R²⁰ are nothydrogen. Any of the aforementioned substituents can be unsubstituted orsubstituted with an alkoxy, an acyloxy, an acylthio, a hydroxy, a haloor a benzyl group.

Alternatively, R¹⁹ and R²⁰, together with the nitrogen atom to whichthey are bonded, form a heterocyclic ring selected from the groupconsisting of:

wherein A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R ishydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branched chain alkyl, aC₃₋₈ cycloalkyl, a substituted or an unsubstituted aryl (such as phenyl,tolyl or the like), or carboxylato and derivatives thereof as furtherdescribed herein, and R⁹ is hydrogen, a C₁₋₆ straight chain alkyl or aC₃₋₆ branched chain alkyl. The aforementioned groups can beunsubstituted or substituted as appropriate.

Exemplary aza crown groups (i.e., where A is N) are 1-aza-12-crown-4,1-aza-15-crown-5, and 1-aza-18-crown-6. Where A is nitrogen, thenitrogen atom, itself, can be substituted, as described, for example, inU.S. patent application Ser. No. 08/475,732.

Further examples include the O²-glycosylated diazeniumdiolates derivedfrom the compounds disclosed in U.S. Pat. No. 5,250,550, and, thus, havethe formula

wherein D is

and wherein R²¹ is the same as the R in the saccharide of Formula Ia andis defined as above, and R¹⁰ and R¹¹, which can be the same ordifferent, can be any suitable group, examples of which includehydrogen, a C₃₋₈ cycloalkyl, a C₁₋₁₂ straight or branched chain alkyl,benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,t-butoxycarbonyl and 2,2,2-trihalo-t-butoxycarbonyl. In Formula IV, f isan integer from 0 to 12.

A preferred O²-glycosylated diazeniumdiolate is one in which, withrespect to Formula Ia, X is N(CH₂CH₂NH₂)₂ and R is fucose or mannose.

The above compounds can be prepared in accordance with methods known tothose of skill in the art. Reagents for glycopyranosylation includeacetobromo-α-galactose and acetobromoglucosamine. Reagents forglycofuranosylation include tribenzyl-α-arabinofuranosyl bromide andbromoacetylxylose.

Oligosaccharides are commercially available from, for example, SigmaChemical Co. (St. Louis, Mo.) and Carbomer Specialty Biochemicals andPolymers (Westborough, Mass.). In addition, oligosaccharides can besynthesized in accordance with well-established procedures, includingchemical and enzymatic preparation, such as those described inPreparative Carbohydrate Chemistry, Stephen Hanessian, ed., MarcelDekker, New York, N.Y. (1997) and Polysaccharides in MedicinalApplications, Severian Dumitriu, ed., Marcel Dekker, New York, N.Y.(1996).

A protected straight- or branched-chain polysaccharide can be activatedtoward reaction with the diazeniumdiolate ion by halogenation of theanomeric terminus, followed by glycosylation of the diazeniumdiolate.Activated disaccharides for generation of O²-glycosylateddiazeniumdiolates include acetobromo-α-maltose and acetobromo-α-lactose.

O²-Glycosylated diazeniumdiolates are useful where molecular signallingand recognition processes, including cell adhesion, involvecarbohydrates. For example, O²-glycosylated diazeniumdiolates arebelieved to be useful in the treatment of infection, such as that due toa parasite (e.g., leishmania), a virus or a bacterium, as well asinflammation and metastasis. In this regard, an O²-glycosylateddiazeniumdiolate can be prepared so as to be directed to amannose-fucose receptor as exemplified in Example 36. It is believedthat the sugar residue, in this instance mannose, protects thediazeniumdiolate. The mannose binds to the mannose-fucose receptor on amacrophage, and the O²-mannosylated diazeniumdiolate is imported intothe cell, where the sugar residue is cleaved, and NO is released.

1-[(2-CARBOXYLATO)PYRROLIDIN-1-YL]DIAZENIUMDIOLATES

With respect to the 1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates,the moiety X of Formula Ia can be

such that the 1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates can bestructurally represented by the formula:

wherein R²² is hydrogen, hydroxyl, OM, wherein M is a cation, halo, orX¹R²³R²⁴, wherein X¹ is N, O or S and, when X¹ is N, R²³ and R²⁴ areindependently a substituted or an unsubstituted C₁₋₂₄ alkyl, C₃₋₂₄cycloalkyl, C₂₋₂₄ olefinic, aryl (such as acridine, anthracene, benzene,benzofuran, benzothiophene, benzoxazole, benzopyrazole, benzothiazole,carbazole, chlorophyll, cinnoline, furan, imidazole, indole,isobenzofuran, isoindole, isoxazole, isothiazole, isoquinoline,naphthalene, oxazole, phenanthrene, phenanthridine, phenothiazine,phenoxazine, phthalimide, phthalazine, phthalocyanine, porphin,pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrocoline, pyrrole, quinolizinium ion, quinoline, quinoxaline,quinazoline, sydnone, tetrazole, thiazole, thiophene, thyroxine,triazine, and triazole), or heterocyclic group, such as glycosyl, andthe like, and when X¹ is O or S, there is no R²⁴ group. Alternatively,when X¹ is nitrogen, R²³ and R²⁴, together with the nitrogen to whichthey are bonded, form a heterocyclic ring, such as a heterocyclic ringselected from the group consisting of:

in which A is N, O or S, w is 1 to 12, y is 1 or 2, z is 1 to 5, R⁸, R⁹,R²⁵ and R²⁶ are hydrogen, a C₁₋₈ straight chain alkyl, a C₃₋₈ branchedchain alkyl, a C₃₋₈ cycloalkyl, or an aryl. The aforementioned groupscan be unsubstituted or substituted as appropriate.

The R substituent on the nitrogen (N−4) can be a hydrogen, a C₁₋₈ alkylgroup, an aryl group, or C(O)-YR²⁷, wherein Y is sulfur or oxygen, ornitrogen and R²⁷ is CH₂OCH₃, vinyl, a C₁₋₉ straight chain alkyl, a C₃₋₆branched chain alkyl, a C₃₋₈ cycloalkyl, polyethylene glycol,polysaccharide, or other polymer, a peptide, or a protein. YR²⁷ can bean activating linker, such as a hydroxy succinimidyl group, for linkageto proteins, peptides, phospholipids, polysaccharides, oligosaccharides,purines, pyrimidines, and biocompatible polymers (i.e., polyethyleneglycol, polylactides, and polycaprolactone). YR²⁷ can be an activatingmoiety for the carbonyl group, making the carbonyl group anelectrophilic site that reacts with nucleophilic functionalities ofoligopeptides, polyamines and proteins. YR²⁷ can cause the carbonylgroup to react with many nucleophiles, and can react with a polymer,such as polyethylene glycol, to form a polymer-bound compound.

Further with respect to the1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates, the moiety R ofFormula Ia can be any covalently bound organic or inorganic moiety,which is other than hydrogen and is a C₁₋₁₂ straight chain or C₃₋₁₂branched chain alkyl, a C₂₋₁₂ straight chain or C₃₋₁₂ branched chainolefinic, a C₁₋₁₂ acyl, sulfonyl, C₃₋₁₂ cycloalkyl, carboxamido, aglycosyl group as described above, an aryl group as described above, ora group of the formula —(CH₂)_(n)—ON═N(O)NR²⁸R²⁹, wherein n is aninteger of 2-8, and R⁸ and R⁹ are independently a C₁₋₁₂ straight chainalkyl, a C₃₋₁₂ branched chain alkyl, a C₁₋₁₂ straight chain or C₃₋₁₂branched chain olefinic, or R²⁸ and R²⁹, together with the nitrogen atomto which they are bonded, form a heterocyclic group, preferably apyrrolidino, piperidino, piperazino or morpholino group. Theaforementioned R groups can be unsubstituted or substituted asappropriate. Preferred substitutions include those made with hydroxy,halo, acyloxy, alkoxy, acylthio, or benzyl.

The above compounds can be prepared in accordance with methods known tothose of skill in the art. For example, see Sanger, Biochem. J. 39:507-515 (1945).

O²-Substituted 1-[(2-carboxylato)pyrrolidin-1-yl]diazeniumdiolates offeradvantages over other diazeniumdiolates in that they are more stable inaqueous solution than the O²-unsubstituted anion and, in many cases,they can be activated for NO release by enzymatic action. Furthermore,if an N-nitroso derivative is formed by net formal cleavage of the N—Ndouble bond of the1-[(2-carboxylato)pyrrolin-1-yl]diazen-1-ium-1,2-diolate, the N-nitrosocompound is noncarcinogenic. Such compounds are believed to beparticularly useful in the treatment of fulminant liver failure,malaria, respiratory problems, impotence, and a variety ofcardiovascular/hematologic disorders.

Polymer Bound Diazeniumdiolates

Another particularly useful embodiment of the present inventioncomprises O²-aryl diazeniumdiolates of Formula I or O²-glycosylateddiazeniumdiolates of Formula Ia, wherein X is a polymer, or wherein anyO²-aryl diazeniumdiolate or O²-glycosylated diazeniumdiolate of thepresent invention is incorporated into a polymeric matrix. PROLI/NO alsocan be polymer bound—through R²⁰ as well as R. Both of these embodimentsresult in the N₂O₂ ⁻ functional group being “bound to the polymer.” By“bound to a polymer,” it is meant that the N₂O₂ ⁻ functional group isassociated with, part of, incorporated with or contained within thepolymeric matrix physically or chemically.

Physical association or bonding of the N₂O₂ ⁻ functional group to thepolymer may be achieved by coprecipitation of the polymer with a nitricoxide/nucleophile complex as well as by covalent bonding of the N₂O₂ ⁻group to the polymer. Chemical bonding of the N₂O₂ ⁻ group to thepolymer may be by, for example, covalent bonding of the nucleophilicmoiety of the nitric oxide/nucleophile adduct to the polymer such thatthe nucleophilic residue to which the N₂O₂ ⁻ group is attached formspart of the polymer, itself, i.e., is in the polymer backbone or isattached to pendant groups on the polymer backbone. The manner in whichthe nitric oxide-releasing N₂O₂ ⁻ functional group is associated with,part of, or incorporated with or contained within, i.e., “bound” to thepolymer is inconsequential to the present invention and all means ofassociation, incorporation and bonding are contemplated herein.

Site-specific application of the polymer-bound adduct compositionenhances the selectivity of action of the nitric-oxide releasing N₂O₂functional group. If N₂O₂ ⁻ functional groups attached to the polymerare necessarily localized, then the effect of their nitric oxide releasewill be concentrated in the tissues with which they are in contact. Ifthe polymer is soluble, selectivity of action can still be arranged, forexample, by linkage to or derivatization of an antibody specific to thetarget tissue. Similarly, linkage of N₂O₂ ⁻ groups to small peptidesthat mimic the recognition sequences of ligands for important receptorsprovides localized nitric oxide release, as would linkage tooligonucleotides capable of site-specific interactions with targetsequences in a nucleic acid.

The O²-diazeniumdiolates of the present invention can be derived fromthe materials disclosed in U.S. Pat. Nos. 5,525,357 (Keefer et al:) and5,405,919 (Keefer et al.), and in U.S. patent application Ser. No.08/419,424 (Smith et al.), each of which is incorporated by reference.Any of a wide variety of polymers can be used in the context of thepresent invention. It is only necessary that the polymer selected isbiologically acceptable. Illustrative of polymers suitable for use inthe present invention are polyolefins, such as polystyrene,polypropylene, polyethylene, polytetrafluorethylene, polyvinyl chloride,polyvinylidene difluoride, and polyethers such as polyethylene glycol,polysaccharides such as dextran, polyesters such aspoly(lactide/glycolide), polyamides such as nylon, polyurethanes,polyethyleneimines, biopolymers such as peptides, proteins,oligonucleotides, antibodies and nucleic acids, starburst dendrimers,polysaccharides, and the like.

In this regard, a polymer containing a diazeniumdiolate can be reactedwith a saccharide, such that the saccharide becomes bound to the N₂O₂ ⁻functional group.

Formation of a diazeniumdiolate from a biopolymer provides abiopolymer-bound diazeniumdiolate composition that can be applied withspecificity to a biological site of interest. Site-specific applicationof the biopolymer-bound diazeniumdiolate enhances the selectivity ofaction of the nitric oxide-releasing diazeniumdiolate, which occursfollowing the cleavage of the O²-aryl or O²-glycosylated bond or the O—Rbond in PROLI/NO (see pg. 33). As with the other polymers disclosedabove, if the diazeniumdiolate attached to the biopolymer is localizedbecause of the inherent properties of the molecule, then the effect ofits nitric oxide release will be concentrated in the tissues with whichthey are in contact. If the biopolymer is soluble, selectivity of actioncan still be arranged, for example, by attachment to or derivatizationof an antibody specific to the target tissue. Similarly, linkage ofdiazeniumdiolate groups to small peptides that mimic the recognitionsequences of ligands for important receptors provides localized nitricoxide release, as would linkage to oligonucleotides capable ofsite-specific interactions with target sequences in a nucleic acid.Other proteins, peptides, polypeptides, nucleic acids andpolysaccharides can be similarly utilized. U.S. Pat. No. 5,405,919(Keefer et al.) and U.S. Pat. No. 5,632,981 (Saavedra et al.), herebyincorporated in their entireties by reference, disclose similarcompounds and manufactures useful in the preparation of thediazeniumdiolates.

By way of illustration, an O²-arylated piperazine diazeniumdiolate canbe covalently attached to a polypeptide containing the IKVAV recognitionsequence, which is important in tumor cell chemotaxis. Through retentionof both the capacity to regenerate NO as an anti-adhesive agent and theaffinity of the IKVAV sequence for tumor cells and/or sites in thevascular and lymphatic systems, where the tumor cells tend to attach,metastasis can be reduced or even prevented. Further, the aryl moietycan be chosen such that it provides additional antitumor cell activity.Substitutions at the N⁴ position of piperazine can be used to link theglycosylated diazeniumdiolate to peptides, polypeptides, proteins,polysaccharides and nucleotides.

It is contemplated that the diazeniumdiolates of the present inventioncan be used to coat prostheses, stents, and medical implants, such asbreast implants, prior to surgical connection to the body as a means ofreducing the risk of solid state carcinogenesis associated therewith.Additionally, the prostheses and implants can be manufactured using adiazeniumdiolate as an integral component of the starting materials.Medical devices incorporating a diazeniumdiolate provide an invaluabletwo-pronged approach to the treatment of many biological disorders,providing useful medical structures that also advantageously providelocal release of NO.

Compositions

As is well-known in the art, nitric oxide and compounds comprising N₂O₂⁻ functional groups can have a wide range of utilities, in part becauseof the multifaceted role of nitric oxide in bioregulatory processes.Accordingly, the present invention also provides a composition,including a pharmaceutical composition, comprising a present inventivediazeniumdiolate. Preferably, the pharmaceutical compositionadditionally comprises a pharmaceutically acceptable carrier.

One skilled in the art will appreciate that suitable methods ofadministering the diazeniumdiolate compositions of the present inventionto an animal, such as a mammal, are available, and, although more thanone route can be used to administer a particular composition, aparticular route can provide a more immediate and more effectivereaction than another route. Pharmaceutically acceptable carriers arealso well-known to those who are skilled in the art. The choice ofcarrier will be determined, in part, both by the particular compositionand by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations of thepharmaceutical compositions of the present invention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the diazeniumdiolate dissolvedin diluents, such as water or saline, (b) capsules, sachets or- tablets,each containing a predetermined amount of the active ingredient, assolids or granules, (c) suspensions in an appropriate liquid, and (d)suitable emulsions. Tablet forms can include one or more of lactose,mannitol, corn starch, potato starch, microcrystalline cellulose,acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, such carriers as are known in the art.

The diazeniumdiolates of the present invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration include aqueous andnon-aqueous solutions, isotonic sterile injection solutions, which cancontain antioxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the animal over a reasonable time frame. The dose will bedetermined by the strength of the particular compositions employed(taking into consideration, at least, the rate of NO evolution, theextent of NO evolution, and the bioactivity of the decompositionproducts derived from the diazeniumdiolates) and the condition of theanimal, as well as the body weight of the animal to be treated. The sizeof the dose also will be determined by the existence, nature, and extentof any adverse side effects that might accompany the administration of aparticular composition. A suitable dosage for internal administration is0.01 to 100 mg/kg per day. A preferred dosage is 0.01 to 35 mg/kg perday. A more preferred dosage is 0.05 to 5 mg/kg per day. A suitableconcentration of O²-aryl diazeniumdiolates in pharmaceuticalcompositions for topical administration is 0.05 to 15% (by weight). Apreferred concentration is from 0.02 to 5%. A more preferredconcentration is from 0.1 to 3%.

Methods of Use

In view of the above, the present invention provides methods of using apresent inventive diazeniumdiolate. In one embodiment, a method oftreating an animal, such as a mammal, with a biological disordertreatable with nitric oxide, is provided. The method comprisesadministering to the animal, e.g., the mammal, an amount of andiazeniumdiolate in accordance with the present invention sufficient totreat the biological disorder in the animal. In this embodiment,“biological disorder” can be any biological disorder, including abiological disorder due to a genetic defect or infection with aninfectious agent, such as a virus, bacterium or parasite, as long as thedisorder is treatable with nitric oxide.

In another embodiment of a method of use, a method is provided fortreating an animal, such as a mammal, for infection with, for example, avirus, a bacterium, or a parasite (e.g., leishmania). The methodcomprises administering to the animal, e.g., the mammal, an amount of adiazeniumdiolate sufficient to treat the infection in the animal.

In one aspect of this embodiment of the invention, a method is providedfor treating an animal, such as a mammal, for infection with, forexample, a virus, such as a retrovirus, in particular HIV, moreparticularly HIV-1, a bacterium, such as a Gram-positive bacterium, or aparasite, such as Giardia, any one of which comprises a zinc fingerprotein that can be inactivated by an O²-aryl diazeniumdiolate. By “zincfinger protein” is meant a protein comprising a short amino acid domaincontaining cysteines alone or cysteine and histidine ligands, both ofwhich coordinate with zinc and interact with nucleic acids (South andSummers, “Zinc Fingers,” Chapter 7, In: Adv. Inorg. Biochem. Ser. 8, pp.199-248 (1990), which is hereby incorporated by reference in itsentirety, including the content of all references cited therein). By“inactivated” is meant partial or complete loss of activity of the zincfinger protein to be inactivated. Such inactivation should not result ininactivation of biologically important zinc finger proteins in theanimal, itself, to such an extent as to compromise unduly the health andwell-being of the animal. The method comprises administering to theanimal, e.g., the mammal, an amount of an O²-aryl diazeniumdiolatesufficient to inactivate the zinc finger protein in said infectiousagent so as to treat the infection in the animal.

The above-described method also can be adapted as a means of treating aplant, plant cell or tissue culture thereof for infection with aninfectious agent, such as a virus, e.g., tobacco streak virus (TSV) oralfalfa mosaic virus (AIMV) (South and Summers (1990), supra; and Sehnkeet al., Virology 168: 48 (1989)).

The methods described herein are useful against zinc fingers comprisingthe motif C—X2—C—X4—H—X4—C (see, e.g., Wain-Hobson et al., Cell 40(1):9-17 (1985)), in which “C” represents cysteine, “H” representshistidine, “X” represents any amino acid, and the numbers “2” and “4”represent the number of “X” amino acids. Such a motif is characteristicof retroviruses, in particular the gag protein of retroviruses.Accordingly, the methods herein are useful against retroviruses, such asHIV, and, in particular, HIV-1 (Rice et al., Nature Medicine 3(3):341-345 (1997); and Rice et al., Reviews in Medical Virology 6: 187-199(1986)), which comprises nucleocapsid p7 proteins (NCp7 proteins) thatinclude two zinc binding domains. Actual and/or potential zinc fingersalso have been identified in, among others, the gene products of the EIAgenomic region of adenoviruses, the large T antigens from simian virus40 (SV40) and polyoma viruses, the UvrA protein in E. coli (Culp et al.,PNAS USA 85: 6450 (1988)), murine leukemia virus (MuLV-F; Green et al.,PNAS USA 86: 4047. (1989)), and bacteriophage proteins (Berg, Science232: 484 (1986)), such as gene 32 protein (G32P) from bacteriophage T4(Giedroc et al., Biochemistry 28: 2410 (1989)). Such proteins can beisolated in accordance with methods known in the art (see referencescited in South and Summers (1990), supra), and the O²-aryldiazeniumdiolates, which can inactivate such zinc finger proteins, canbe identified in accordance, for example, with the zinc finger assaydescribed herein and in Rice et al., J. Med. Chem. 39: 3606-3616 (1996).

To the extent that steroid hormone receptors comprise zinc fingers withmotifs comprising 4 or 5 cysteines, an O²-aryl diazeniumdiolate can beused to modulate steroid hormone activity in an animal, such as amammal. Accordingly, the present invention also provides a method ofmodulating steroid hormone activity in an animal, such as a mammal,which is in need of modulation of steroid hormone activity and whichcomprises a steroid hormone receptor protein comprising a zinc fingerthat can be inactivated by an O²-aryl diazeniumdiolate. The methodcomprises administering to the animal, e.g., the mammal, an amount of anO² -aryl diazeniumdiolate sufficient to inactivate the steroid hormonereceptor protein so as to modulate steroid hormone activity in theanimal.

In yet another embodiment, a method for treating an animal, such as amammal, for cancer and metastasis thereof is provided. The methodcomprises administering to the animal, e.g., the mammal, an amount ofdiazeniumdiolate sufficient to prevent the growth or metastasis of thecancer in the animal.

In one aspect of this embodiment, a method for treating an animal, suchas a mammal, for cancer is provided, wherein the cancer is due, at leastin part, directly or indirectly, to the activity of a zinc fingerprotein that can be inactivated by an O²-aryl diazeniumdiolate. Themethod comprises administering to the animal, e.g., the mammal, anamount of O²-aryl diazeniumdiolate sufficient to inactivate the zincfinger protein so as to treat the cancer in the animal (Rice et al.,PNAS 89: 7703-7707 (1992)), i.e., prevent the growth or metastasis ofthe cancer in the animal.

In still yet another embodiment, a method is provided for treating ananimal, such as a mammal, for cancer, wherein the cancer is resistant totreatment with a chemotherapeutic agent (see, e.g., Kelley et al.,Biochem. J. 304: 843-848 (1994)), in particular a DNA damaging agent,such as an alkylating agent or an oxidizing agent, due, for example, tothe action of an enzyme that adversely affects the activity of thechemotherapeutic agent. The method comprises administering to theanimal, e.g., the mammal, an amount of an O²-aryl diazeniumdiolatesufficient to render the cancer in the animal susceptible to treatmentwith the chemotherapeutic agent. Accordingly, such a method can be usedas an adjunct therapy to chemotherapy as needed.

For example, certain O²-aryl diazeniumdiolates can be synthesized to fitinto the active site of glutathione S-transferase, specificallyisoenzyme π (see, e.g., Ji et al., Biochemistry 32(49): 12949-12954(1993); and Ji et al., Biochemistry 36: 9690-9702 (1997)). Accordingly,inversible consumption or glutathione from the active site ofglutathione S-transferase-7 with an O²-aryl diazeniumdiolate couldprevent the enzyme from detoxifying a variety of xenobiotic compounds,such as chemotherapeutic drugs, especially alkylating agents, such aschlorambucil, melphalan and hepsulfam, and other DNA-damaging agents,such as agents that induce electrophilic attack or oxidization, byenzymatic conjugation of the compound with glutathione (see, e.g.,Morgan et al., Cancer Chemother. Pharmacol. 37: 363-370 (1996)). Thismethod also has applicability to screening drug-resistant cancer celllines in vitro.

In another embodiment, a method is provided for treating an inanimateobject for the presence of a potentially infectious virus, bacterium, orparasite. The method comprises contacting the inanimate object with anamount of a present inventive diazeniumdiolate sufficient to reduce thepresence of the potentially infectious virus, bacterium or parasite. By“potentially infectious” is meant the capability of infecting an animal,such as a mammal.

In one aspect of this embodiment, a method is provided for reducing onan inanimate object the presence of a potentially infectious agent, suchas a virus, a bacterium, or a parasite, any one of which comprises azinc finger protein that can be inactivated by an O²-aryldiazeniumdiolate. The method comprises contacting the inanimate objectwith an amount of an O²-aryl diazeniumdiolate sufficient to inactivatethe zinc finger protein so as to reduce the presence of the potentiallyinfectious agent, e.g., virus, bacterium or parasite, on the inanimateobject. By “potentially infectious” is meant the capability of infectingan animal, such as a mammal, directly or indirectly.

EXAMPLES

The following examples further illustrate the present invention and, ofcourse, should not be construed as in any way limiting its scope. Withrespect to the following examples, NO was obtained from Matheson GasProducts (Montgomeryville, Pa.), β- and α-glycosidases and porcine liveresterase were obtained from Sigma Chemical Co. (St. Louis, Mo.),polyurethane (Tecoflex) was obtained from Thermedics Inc. (Woburn,Mass.), and glucose and mannose were obtained from Aldrich Chemical Co.(Milwaukee, Wis.). Proton NMR spectra were recorded with a 300 MHzVarian Unity Plus or a Varian XL-200 NMR spectrometer. Spectra wereobtained in deuterochloroform for covalent compounds and in D₂O forsalts. Chemical shifts are reported in parts per million (ppm) downfieldfrom TMS. Low and high resolution mass spectral (MS) measurements werecarried out on a VG-Micromass Model 7070 spectrometer. Unless otherwiseindicated, MS data were collected in the electron impact mode withsample introduction via direct probe. Ultraviolet (UV) spectra were runas solutions in water or 0.01 M NaOH on an HP 8451A Diode Arrayspectrophotometer. Glutathione S-transferase kinetics were monitored bymeasuring the change in UV absorbance at 380 nm with a Beckman DU 640spectrophotometer. Chemiluminescence measurements were done on a ThermalEnergy Analyzer Model 502A instrument (Thermedics, Inc., Woburn, Mass.).Elemental analyses were performed by Atlantic Microlab Inc.

EXAMPLE 1

This Example illustrates the preparation of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

A solution of 1.67 g (11 mmol) of sodium diethylaminodiazeniumdiolate in20 ml of 5% aqueous sodium bicarbonate was cooled to 0° C. undernitrogen. A solution of 1.3 ml (0.01 mol) of 2,4-dinitrofluorobenzene in10 ml of t-butyl alcohol was added slowly. A precipitate formed uponaddition. The mixture was allowed to warm up to room temperaturegradually, then stirred overnight. The product was extracted withdichloromethane and subsequently washed with cold dilute hydrochloricacid followed by sodium bicarbonate solution. The organic layer wasdried over sodium sulfate, filtered through a layer of magnesiumsulfate, and evaporated under vacuum to give 1.3 g of a red oil, whichcrystallized on standing. Recrystallization from ethanol gaveyellow-orange needles: m.p. 76-7° C.; NMR δ 1.25 (t, 6H), 3.58 (q, 4H),7.68 (d, 1H), 8.44 (m, 2H), 8.89 (m, 1H) ; UV (ethanol) λ_(max) (ε) 218(17.4 mM⁻¹ cm¹⁻) and 302 (15.6 mM⁻¹ cm⁻¹) nm; MS, exact mass, calculatedfor C₁₀H₁₃N₅O₆: (M+) 299.0865; measured M⁺ 299.08658. Analysis, C, H, N,calculated for: C₁₀H₁₃N₅O₆: C 40.13%, H 4.35%, N 23.41%. Found: C40.21%, H 4.43%, N 23.37%.

EXAMPLE 2

This Example illustrates the regeneration of the anionicdiazeniumdiolate from its O²-aryl substituted form(O²-(2,4-dinitrophenyl) 1-(N,N-diethyamino)diazen-1-ium-1,2-diolate).

A solution of 85 mg (0.28 mmol) of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, prepared as in Example 1,in 1 ml of ether was cooled to −4° C. and treated with 1 ml ofdiethylamine. The solution was kept at −4° C. for 1 hr, giving aprecipitate. The solid was collected by filtration. The filtrate wasconcentrated and analyzed by NMR; the residue proved to be identical toan authentic sample of 2,4-dinitro-N,N-diethylaniline. The precipitatewas washed with petroleum ether and dried under N₂ to give 5.4 mg ofproduct having λ_(max) 250 nm; NMR (D₂O) δ 0.96 (t, 6H), 1.28(t, 6H),2.94 (q, 4H), 3.08 (q, 4H). This product proved to be identical to anauthentic sample of diethylammonium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

EXAMPLE 3

This Example illustrates the chemical cleavage of the O²-aryl bond of anO²-aryl diazeniumdiolate mediated by sodium methoxide.

A solution of 16 mg (0.064 mmol) of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate in 1 ml of ether wastreated with 29 μl of 25% sodium methoxide in methanol (0.14 mmol) andallowed to stand at −4° C. for 2 hr. The solid precipitate was collectedby filtration, washed with ether and dried under vacuum to yield 4 mg ofa solid identical to an authentic sample of1-(N,N-diethylamino)diazen-1-ium-1,2-diolate sodium salt.

EXAMPLE 4

This Example illustrates the kinetics of reaction ofO²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate withsodium methoxide in methanol. The kinetics of this reaction show therate of conversion of O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate to1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion in alkaline ornucleophilic environments.

An excess of NaOMe was used in the reactions; aliquots were collected atintervals and quenched with 0.1 N HCl in methanol. The disappearance ofO²(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate,monitored by HPLC, was found to fit the first-order rate equation. Thiswas determined by plotting log[O²-(2,4-dinitrophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate] vs. time to find k_(obs')at four different concentrations of NaOMe. Similarly, the second-orderrate constant (7.87 M^('11) min⁻¹) was determined by plotting log kobsVs. log[NaOMe].

EXAMPLE 5

This Example illustrates the preparation of O²-(2,4-dinitrophenyl)1-(N-isopropylamino)diazen-1-ium-1,2-diolate.

A solution of 84 mg (0.597 mmol) of sodium1-(N-isopropylamino)diazen-1-ium-1,2-diolate in 1 ml of 5% sodiumbicarbonate was cooled to 0° C. and 69 mg (0.55 mmol) of2,4-dinitrofluorobenzene was added. The ice bath was removed, themixture was allowed to stir at room temperature overnight, and then themixture was extracted with dichloromethane. The extract was dried oversodium sulfate, filtered and evaporated in vacuo to give 86 mg of afilm, which crystallized on standing: m.p. 92-93° C. NMR δ 1.39 (d, 6H),3.99 (septet, 1H), 6.93 (d, 1 H), 8.27 (dd, 1H), 8.5 (b, 1H), 9.15 (d,1H).

EXAMPLE 6

This Example illustrates the synthesis of pyrrolidinium1-[pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

A solution of 36 g (0.507 mol) of pyrrolidine in 50 ml of ether and 25ml of acetonitrile was placed in a 500 ml Parr bottle, degassed andcharged with 40 psi of nitric oxide. The reactor was cooled to −80° C.The pressure was maintained at 40 psi. After 4 hr, the pressure wasreleased, and the crystalline product was collected by filtration in afritted glass funnel and then washed with cold ether under an atmosphereof nitrogen. The material was dried in a vacuum desiccator at 1 mm Hgand 25° C. for 3 hr to give 23 g (45%) of white needles: m.p. 68 70° C.Analysis C,H,N: Calculated for C₈H18N₄O₂: C 47.51%, H 8.97%, N 27.70%;Found, C 47.62%, H 9.04%, N 27.46%.

The pyrrolidinium salt was converted to the more stable sodium salt forsubsequent O²-arylations by treatment with 10 N NaOH to promote cationexchange. It was then flooded with ether. The product was collected byfiltration.

EXAMPLE 7

This Example gives an alternate method of preparing the sodium salt ofthe 1-(pyrrolidin-1-yl)diazen-1-ium1,2-diolate presented in Example 6.

A solution of 28.2 g (0.397 mol) of pyrrolidine in 100 ml ofacetonitrile and 100 ml of ether was mixed with 94 ml (0.4 mol) of 25%sodium methoxide in methanol. The resulting solution was flushed withnitrogen then charged with 40 psi of NO and stirred at room temperaturefor two days forming a thick precipitate. (The precipitate had begun toform within 1 hr of exposure to NO.) The pressure was released and theproduct was collected by filtration. The product was washed with etherand dried under vacuum to give 32.1 g (54%) of a white powder: UV (0.01N NaOH) λ_(max) (ε), 252 nm (8.84 mM⁻¹ cm⁻¹); t_(½) 8.5 sec at 25° C.and 2.8 sec at 37° C. in pH 7.4 phosphate buffer; NMR (D₂O) δ 1.91 (m,4H), 3.22 (m, 4H).

EXAMPLE 8

This Example illustrates the preparation of O²-(2,4-dinitrophenyl)1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate.

A solution of 556 mg (3.63 mmol) of sodium1(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate in 10 ml of 5% aqueous sodiumbicarbonate was cooled to 0° C. A solution of 456 μl (3.63 mmol) of2,4-dinitrofluorobenzene in 2 ml of t-butyl alcohol was added and theresulting mixture was stirred at room temperature overnight. Theyellow-orange precipitate was collected by filtration, washed withwater, and dried to give 758 mg of product, which was recrystallizedfrom ethanol: m.p. 94-95° C.; NMR, δ 2.04 (m, 4H), 3.35 (m, 4H), 6.90(d, 1H), 8.20 (dd, 1H), 8.67 (d, 1H); MS, m/z(%), 297 (M⁺, 1), 220(100), 237 (30), 190 (94), 180 (15), 162 (10), 149 (26), 130 (20), 100(95), 70 (24), 63 (35), 56 (18). Exact Mass: calculated for C₁₀H₁₁N₅O₆(M⁺)297.0708; measured 297.0709.

EXAMPLE 9

This Example illustrates the preparation of sodium1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate.

A solution of 20 g (0.126 mol) of N-carboethoxypiperazine in 60 ml ofmethanol was placed in a Parr bottle. The solution was treated with 27.4ml (0.126 mol) of 25% sodium methoxide in methanol; the system wasevacuated, charged with 40 psi of nitric oxide and kept at 25° C. for 48hr. The white crystalline product was collected by filtration and washedwith cold methanol as well as with copious amounts of ether. The productwas dried under vacuum to give a 14.5 g (48%) yield of sodium1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate: m.p.184-5° C.; UV (0.01 N NaOH) λ_(max) (ε) 252 nm (10.4 mM⁻¹ cm⁻¹); NMR(D₂O) δ 1.25 (t, 3H), 2.15 (q, 2H) 3.11 (m, 4H), 3.68 (m, 4H). Analcalcd. for C₆H₁₃N₄O₄Na: C 35.00%, H 5.42%, N 23.33%, Na 9.58%. Found: C34.87%, H 5.53%, N 23.26%, Na 9.69%.

The half-life of this compound at pH 7 and 25° C. was assessed at 5 min.This measurement was based on the loss of the 252 nm chromophore in theultraviolet spectrum.

EXAMPLE 10

This Example illustrates the preparation of O²-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate.

A solution of 1.073 g (0.0045 mol) of sodium1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate in 10 ml of5% sodium bicarbonate was cooled at 0° C. under nitrogen. A partialsolution of 0.89 ml (0.0044 mol) of 2,4-dinitrofluorobenzene in 10 ml oft-butyl alcohol was added. A precipitate formed upon addition; themixture was allowed to stir at room temperature for 4 hr. The productwas extracted with dichloromethane. The extracts were washed with water,dried over sodium sulfate and filtered through a layer of anhydrousmagnesium sulfate. Evaporation of the solvent gave an orange glass whichcrystallized on standing. The product was recrystallized fromethanol:dichloromethane to give 1.3 g (76%) of analytically purematerial: m.p. 140-141° C.; NMR δ 1.32 (t, 3H), 3.63 (m, 4H), 3.74 (m,4H), 4.19 (q, 2H), 7.66 (d, 1H), 8.48 (q, 1H), 8.88 (d, 1H); UV (H₂O)λ_(max) (ε) 210 nm (13.3 mM⁻¹ cm⁻¹), 300 nm (12 mM⁻¹ cm⁻¹). Anal calcd.for C₁₃H₁₆N₆O₈: C 40.61%, H 4.20%, N 21.87%; Found: C 40.74%, H 4.13%, N21.98%.

EXAMPLE 11

This Example illustrates the preparation of O²-(2-chloropyrimidin-4-yl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

A solution of 600 mg (4 mmol) of 2,4-dichloropyrimidine in 2 ml ofdimethylsulfoxide and 5 ml of tetrahydrofuran was added via syringe to aslurry of 678 mg (4.37 mmol) of sodium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate in 5 ml of tetrahydrofuranat room temperature under nitrogen and the resulting mixture was stirredfor 72 hr. Five (5) ml of ether was added to the mixture. After washingwith water, the organic layer was dried over sodium sulfate, filteredthrough a layer of magnesium sulfate, and evaporated to give 679 mg ofan oil which crystallized at −20° C. This material was recrystallizedfrom ether-petroleum ether: m.p. 37-38° C.; NMR δ 1.25 (t, 6H), 3.56 (q,4H), 7.00 (d, 1H), 8.50 (d,1H); UV, λ_(max) (ε) 268 nm (9.3 mM⁻¹ cm⁻¹).Analysis C, H, N: Calculated for C₈H₁₂N₅O₂Cl: 39.11%, H 4.92%, N 28.51%,Cl 14.43%; Found: C 38.96%, H 4.96%, N 28.35%, Cl 14.60%.

EXAMPLE 12

This Example illustrates the preparation O²-(2-chloropyrimidin-1-yl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate.

A solution of 262 mg (1.76 mmol) of 2,4-dichloropyrimidine in 3 ml ofdimethylsulfoxide was added to a slurry of 424 mg (1.76 mmol) of sodium1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate in 10 ml oftetrahydrofuran at room temperature under nitrogen and stirred for 72hr. The resulting homogeneous solution was treated with 100 ml of water.The precipitate was collected by filtration and dried under vacuum togive 300 mg of product: m.p. 136-137° C.; NMR δ 1.29 (t, 3H), 3.69 (m,4H), 3.71 (m, 4H), 4.18 (q, 2 H), 6. 99 (d, 1 H), 8.52 (d, 1 H); (UV)λ_(max) (ε) 270 nm (4.1 mM⁻¹ cm⁻¹).

This compound undergoes nucleophilic substitution with methoxide todisplace the chlorine atom at the C2 position and the diazeniumdiolateat the C4 position to give 2,4-dimethoxypyrimidine.

EXAMPLE 13

This Example describes the synthesis of the following compounds:

General synthesis of compounds 1 through 5: A 1 M solution of sodium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate in dimethylsulfoxide wasstirred at 5° C. under nitrogen. A 1 M solution containing 0.95 molarequivalents of the arylating agent in tetrahydrofuran was injectedthrough a septum. The reaction mixture was allowed to warm up to roomtemperature, stirred overnight, quenched with ice-water and extractedwith ether. The ether was washed with water, dried over sodium sulfate,filtered through a layer of maganesium sulfate and concentrated on arotary evaporator. The methods of purification varied with eachpreparation and are described with the individual compounds below.(Note: Compounds 1 through 5 are selected products from O²-aryl compoundlibraries built using solution phase synthetic methods in parallelfashion). NMR spectra were run in CDCl₃.

O²-(2-Nitro-4-trifluoromethylphenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, 1: Arylation was carriedout with 4-fluoro-3-nitrobenzotrifluoride. Purification of the productwas carried out on preparative HPLC using a 1 inch C-18 column elutedwith 20% aqueous acetonitrile with a solvent gradient to 50%acetonitrile:50% water. A 42% yield of product was obtained as an oil:NMR δ 1.23 (t, 6H), 3.50 (q,4 H), 7.66 (d, 1 H), 7.82 (d, 1 H), 8.28 (s,1 H).

O²-(2-Nitro-4-carboxylatophenyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, 2: 4-Fluoro-3-nitrobenzoicacid was used in this preparation. Purification of the product wascarried out on a Biotage Flash 40 system with a 4.0×15.0 cm KP-Silcolumn. The system was eluted with 5:1 dichloromethane:ethyl acetate at15 psi of air at a rate of elution of 25 ml/min to give a 22% yield ofproduct: mp 115-6° C.; NMR δ 1.22 (t, 6 H), 3.33 (q, 4 H), 7.06 (d, 1H), 8.03 (dd, 1 H), 8.37 (m, 1 H).

O²-(5-Nitropyrid-2-yl)1-(N,N-diethyl)diazen-1-ium-1,2-diolate, 3: Theproduct of reaction with 2-bromo-5-nitropyridine was recrystallized fromether:ethanol to give pure 3. in 62% yield: mp 77-8° C.; NMR δ 1.24 (t,6 H), 3.53 (q, 4 H), 7.21 (dd, 1 H), 8.52 (dd, 1 H), 9.17 (dd, 1 H).Analysis C,H,N: Calculated for C₉H₁₃N₅O₄: C 42.35%, H 5.13%, N 27.44%,Found: C 42.46%, H 5.14%, N 27.52%.

O²(3,5-Dinitropyrid-2-yl)1-(N,N-diethyl)diazen-1-ium-1,2-diolate, 4:Anylation was effected with 2-chloro-3,5-dinitropyridine as described inthe general procedure. The crude product was recrystallized fromether:petroleum ether to give 4 in 33% yield: mp 56-7° C.; NMR δ 1.28 (t6 H), 3.57 (q,4 H), 8.81 (d, 1 H), 9.10 (d, 1 H).

O²-(3-Nitropyrid-2-yl)l-(N,N-diethyl)diazen-1-ium-1,2-diolate, 5:2-Chloro-3-nitropyridine was used in this reaction. The crude productwas purified on a Flash 40 system using a 4.0×7.0 cm KP-Sil columneluted with 100% dichloromethane to give a 52% yield of product as aviscous oil: NMR δ 1.25 (t, 6 H), 3.55 (q, 4 H), 7.26 (m, 1 H), 8.48 (m,2 H).

EXAMPLE 14

This Example illustrates the preparation of O²-vinyl1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate(V-PROLI/NO).

To 3.56 g (9.2 mmol) of O²-(2-bromoethyl)1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate 2-bromoethylester was added 10 ml of 10 N sodium hydroxide solution.

The two-phase mixture was stirred at 25° C., whereupon the compoundgradually dissolved in the aqueous layer. After stirring overnight, theUV of the reaction mixture exhibited an absorption maximum at 266 nm(starting material absorbed at 252 nm), indicating the formation of avinyl group.

The solution was cooled to 0° C. and carefully acidified to pH 4 by theslow addition of 10% hydrochloric acid. Care must be taken to keep thesolution cold while acid is added. The acidic solution was extractedwith ethyl acetate, dried over sodium sulfate and filtered through alayer of magnesium sulfate. Evaporation of the solvent gave 1.4 g of anoil. Purification was carried out on a Flash 40 System (Biotage) using a4.0×7.0 cm KP-Sil column and 2:1 ethyl acetate:cyclohexane as theeluant:ir (film) 3163, 2987, 1734, 1630, 1490 cm⁻¹; NMR (CDCl₃) δ2.06-2.3 (m,4H), 3.62 (m,2H), 4.47 (q,1H), 4.77 (ABq, 1H), 5.02 (ABq,1H), 6.75 (q,1H); UV λ_(max) (ε) 266 nm(6.3 mM⁻¹ cm⁻¹); MS,m/z (%) 201(M⁺, 5), 176(10), 150(49), 145(27), 114(9) 99(45), 70(99.9), 69(57),68(45).

EXAMPLE 15

This Example illustrates the regeneration of NO fromO²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate inthe presence, but not the absence, of glutathione.

A solution containing 1 mM glutathione (GSH) in 10 mM phosphate bufferwas degassed by purging with argon for 10 min, whereupon a 3 ml aliquotwas mixed with 3 μl of a dioxane solution that was 2 mM inO²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate. NOrelease was monitored by chemiluminescence while the mixture was held at37° C. After a brief lag time, peak nitric oxide generation was observedat approximately 15 minutes after the reaction was initiated andcontinued at readily detectable levels for approximately 100 minutes.Total NO generation during the first 112 min was approximately 9 nmol.Assuming that 2 nmol of NO is generated per mol ofO2-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate,this 9 nmol corresponds to roughly 75% of the theoretical yield.

When the reaction was repeated as above but with exclusion of the GSH,no NO generation was observed. The nucleophilic glutathione reacted withthe O²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolateto produce NO according to the equation shown below.

This example is illustrative of the ability of some of the O²-aryldiazeniumdiolate compounds of the present invention to undergonucleophilic substitution by nucleophilic side-chains of amino acidssuch as cysteine, which are often found in the active sites of enzymes.The result of such nucleophilic substitution is the generation of anaryl derivative of the displacing amino acid residue and adiazeniumdiolate capable of producing NO, through a predictable,first-order reaction.

O²-(2,4-Dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate andglutathione were also assayed in the presence and absence of glutathioneS-transferase. Assays were conducted in a thermostated cell compartmentat 25° C., using 0.1 M phosphate buffer at pH 7.4, with a final volumeof 3 ml. The concentration of the enzyme was 0.7 μg/ml, whereas that ofglutathione was 1.4 mM. The concentration of diazeniumdiolate was variedfrom 50-100 μM. Using the integrated form of the Henri-Michaelis-Mentenequation, K_(m) was found to be 46.3 μM and V_(max) was found to be 0.89μM min⁻¹.

EXAMPLE 16

This Example illustrates a route of synthesis which is useful in theproduction of diazeniumdiolated nucleotides, nucleosides, and nucleicacids and further illustrates a route to synthesis of O²-aryldiazeniumdiolates, which comprises converting an amino group to adiazonium group, followed by reaction of the diazonium group with adiazeniumdiolate.

2′-Deoxycytidine is reacted with nitric oxide in the presence of asuitable 1-electron oxidant which results in the conversion of the aminogroup of the cytidine into a diazonium group while reducing the oxidantand producing hydroxide ion. The resulting diazotized (i.e., diazoniumderivatized) pyrimidine is then reacted with1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion, as described in theprevious examples, to generate a diazeniumdiolated 2′-deoxyuridinederivative. This diazeniumdiolated 2′-deoxyuridine derivative can bereacted with strong nucleophiles (e.g., hydroxide ions). This willresult in the regeneration of1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion plus 2′-deoxyuridine.This regenerated 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate ion willgenerate NO in a predictable, first-order reaction. This Exampledemonstrates a basis for a mechanism that is suitable for targetingnitric oxide to a particular site of a mammalian body, so that thespecificity of NO action can be increased.

EXAMPLE 17

This example demonstrates the ability of an O²-aryl diazeniumdiolate toinactivate a zinc finger protein by zinc ejection.

Samples of recombinant nucleocapsid protein p7 (p7NC) from HIV-1 (L. O.Arthur, AIDS Vaccine Program, NCI-FCRDC, Frederick, Md.) were preparedat μg/ml in 10 mM sodium phosphate buffer (pH=7.0) and treated with 25μmol of an O²-aryl diazeniumdiolate in a total volume of 1.0 ml. Atvarious time intervals, as shown in FIG. 1, which is a graph of Trp37fluorescence (RFU) versus time (min), the samples were diluted 1/10 in10 mM sodium phosphate buffer (pH=7.0) to prevent introduction of anyartifactual quenching effects and the fluorescence intensity of thetryptophan residue (Trp37) in the C-terminal zinc finger of p7NC in eachsample was determined as previously described (Rice et al., Int.Antiviral News 3: 87-89 (1995)). The excitation and emission wavelengthsutilized with a Shimadzu RF5000 spectrofluorimeter were 280 and 351 nm,respectively. The results are shown in FIG. 1, in which ◯ represents thenegative control, i.e., no drug, □ represents the positive control,i.e., 642151 (see Rice et al. (1997), supra), ▪ represents the compoundof Example 1 (LK1), ♦ represents the compound of Example 8 (LK2), ▴represents the compound of Example 5 (LK3),  represents the compound ofExample 10 (LK4), and x represents the compound of Example 11 (LK5). Theresults indicate that an O²-aryl diazeniumdiolate can eject zinc from azinc finger protein.

EXAMPLE 18

This example demonstrates the anti-HIV activity of O²-aryldiazeniumdiolates.

The tumor cell line of T4 lymphocytes designated CEM-SS was grown in asynthetic medium with fetal bovine serum (Rice et al., Advances inPharmacol. 33: 389-438 (1995)). O²-aryl diazeniumdiolates wereadministered to HIV-1-infected and uninfected CEM-SS cells atconcentrations ranging from 10^(−3.5) to 10^(−7.0) M in accordance withthe XTT-based cell viability assay of the National Cancer Institute(see, e.g., Rice et al. (1995), supra).

After exposure of CEM-SS cells to the compounds, the percentage ofT-cell viability was assessed. The viability of HIV-1-infected CEM-SScells, which were contacted with a subtoxic concentration of any one ofthe above-described O²-aryl diazeniumdiolates, was substantiallyincreased in comparison to untreated cells. Compounds 1-3 from Example13 were especially effective.

EXAMPLE 19

This example describes the preparation of disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

A solution of 10 g (0.087 mol) of L-proline in 39 ml (0.18 mol) of 25%sodium methoxide in methanol, 20 ml of methanol and 40 ml of ether wasdegassed and exposed to 40 psi of nitric oxide for 20 hr. The pressurewas released and the solid residue was collected by filtration, washedwith ether and dried under vacuum to give 17 g of a white solid: mp 250°C. (dec.); UV (0.01 N NaOH) λ_(max) (ε) 252 nm (8.4 mM⁻¹ cm⁻¹); NMR(D₂O) δ 1.71 (m, 1H), 1.91 (m, 2H), 2.27 (m, 1H), 3.27-3.43 (m, 2H),4.04 (m, 1H) (a methanol singlet at 3.34 is also observed); ¹³C NMR,24.45 ppm, 30.97, 48.73 (methanol), 54.95, 67.70, 182.75.

Anal. C,H,N: Calculated for C₅H₇N₃O₄Na₂.CH₃OH, C 28.69%, H 4.41%, N16.73%, Na 18.30%; Found C 28.65%, H 3.99%, N 16.74%, Na 18.04%.

EXAMPLE 20

This example describes the preparation of O²-methyl1-[(2-carboxylato)pyrrolidin-1-yl)diazen-1-ium-1,2-diolate methyl ester.

Disodium 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate(methanol solvate, FW 251; 6.8 g; 0.027 mol) was placed in a 300 ml3-neck flask and cooled to −20° C. Cold methanol (−20° C.; 200 ml) wasadded to the solid while stirring to give a homogeneous solution, whichwas cooled further to −35° C. A solution of 9.5 ml (0.1 mol) ofdimethylsulfate in 25 ml of ether was added dropwise over a 15 minperiod. The reaction mixture was then allowed to warm to roomtemperature gradually and stirred for an additional 4 hr. The progressof the reaction was monitored on silica gel TLC using 10:1dichloromethane:ethyl acetate as the eluant. The reaction mixture wasfiltered, the methanol was removed on a rotary evaporator, and theresidue was extracted with dichloromethane. The solution was washed withaqueous sodium bicarbonate, dried over sodium sulfate and filteredthrough a layer of magnesium sulfate. Evaporation of the solvent gave anoil, which crystallized on standing. Recrystallization fromether:petroleum ether gave 945 mg (18%) of an analytically pure sample:mp 62-63° C.; UV (0.01 N NaOH), λ_(max) (ε) 252 nm (6.79 mM⁻¹ cm⁻¹); NMRδ 2.05 (m, 3H), 2.30 (m, 1H), 3.65 (m, 1H), 3.75 (s, 3H), 3.83 (m, 1H),3.96 (s, 3H), 4.55 (m, 1H); MS m/z (%) 203 (M⁺, 6), 188 (20), 58 (35),120 (22), 99 (100), 95 (34), 69 (36), 59 (24); exact mass calculated forC₇H₁₃N₃O₄ (M⁺) 203.0906, found (M⁺) 203.0906.

Anal. C,H,N: Calculated for C₇H₁₃N₃O₄, C 41.38%, H 6.45%, N 20.68%:Found C 41.48%, H 6.43%, N 20.59%.

EXAMPLE 21

This example describes the preparation of O²-(N,N-dimethylsulfamoyl)1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate.

A solution of 1.08 ml (0.01 mol) of N,N-dimethylsulfamoyl chloride in 5ml of tetrahydrofuran was added dropwise to a cold (0° C.) solution of1.57 g (0.0062 mol) of disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate in 25 ml of0.1 N NaOH in saline solution. The reaction mixture was allowed to warmup to room temperature and stirred overnight. The aqueous layer wasextracted with dichloromethane and the organic layer was dried overanhydrous sodium sulfate. The aqueous layer showed no significant UVabsorption after extraction and, thus, indicated that the extractionproducts were devoid of diazeniumdiolate. The organic layer was filteredthrough a layer of magnesium sulfate and the solvent was removed on arotary evaporator to give 989 mg of a pale yellow oil, which waschromatographed on silica gel using 5:1 dichloromethane:ethyl acetate asthe eluant. The fractions containing the desired product were combinedand concentrated under vacuum to give a solid, which was recrystallizedfrom ether-petroleum ether: mp 97-98° C.; UV (0.01 N NaOH) λ_(max) (ε)266 nm (8.05 mM⁻¹ cm⁻¹); NMR δ 2.16 (m, 3H), 2.40 (m, 1H), 3.01 (s, 6H),3.83 (m, 1H), 3.94 (m, 1H), 4.69 (q, 1H), 6.80 (b, 1H).

Anal. C,H,N,S: Calculated for C₇H₁₄N₄SO₆, C 29.79%, H 5.00%, N 19.85%, S11.36%: Found C 29.93%, H 5.09%, N 19.76%, S 11.27%.

EXAMPLE 22

This example describes the preparation of O²-methoxymethyl1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate methoxymethylester.

A slurry of 485 mg (1.93 mmol) of disodium1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate in 20 ml ofanhydrous tetrahydrofuran was cooled to 0° C. under a nitrogenatmosphere. Triethylamine (0.5 ml) was added to the cold solutionfollowed by the slow addition of 360 mg (4.45 mmol) ofchloromethylmethyl ether and a subsequent dropwise addition of 0.5 ml ofmethanol. The solution was then stirred in the cold for 1.5 hr. Thereaction mixture was allowed to warm up to room temperature and stirredunder nitrogen for an additional 1.5 hr. The reaction was quenched withcrushed ice, whereupon the solvent was removed on a rotary evaporatorand the residue was extracted with dichloromethane. The organic phasewas washed with water, dried over sodium sulfate, filtered throughmagnesium sulfate and evaporated in vacuo to give 330 mg of a yellowoil, which was purified on a silica gel column with 5:1dichloromethane:ethyl acetate as the eluant: UV (H₂O) λ_(max) (ε) 250 nm(8.58 mM⁻¹ cm⁻¹); NMR δ 2.09 (m, 3H), 2.35 (m, 1H), 3.48 (s, H), 3.71(m, 2H), 3.90 (m, 1H), 4.61 (dd, 1H), 5.17 (ab q, 2H), 5.31 (ab q, 2H).

Anal. C,H,N: Calculated for C₉H₁₇N₃O₆: C 41.06%, H 6.51%, N 15.96 %:Found C 40.87%, H 6.53%, N 15.76%.

EXAMPLE 23

This example describes the preparation of O²-(2-bromoethyl)1-((2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate 2-bromoethylester.

A solution of 20 ml (0.28 mol) of bromoethanol in 50 ml ofdichloromethane was cooled to 0° C. and 11.25 ml (0.28 mol) of sulfurylchloride in 50 ml of dichloromethane was added dropwise to the solution.The resulting solution was kept at 4° C. for 72 hr. The solution waswashed with cold 10% NaOH until the washings tested distinctly basic.The organic layer was dried over sodium sulfate, filtered through alayer of magnesium sulfate and concentrated on a rotary evaporator. Theresulting crude product (2-bromoethoxysulfonyl chloride, BrCH₂CH₂OSO₂Cl)was vacuum-distilled to give 35 g (56%) of a colorless oil: bp 73-75° C.at 1.5 mmHg; NMR δ 3.64 (t, 2H), 4.752 (t, 2H); MS m/z (%) 221 (M⁺, 1),143 (10), 129 (25), 106 (100), 93 (62). Analysis C,H,N,S,X: Calculatedfor C₂H₄SO₃ClBr: C 10.75%, H 1.80%, S 14.35%, total halogen as Br 71.52%and as Cl 31.72%; Found: C 10.82% H 1.80%, S 14.35%, total halogen as Br71.63% and as Cl 31.78%.

Disodium 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate(4.86 g; 0.0194 mol) was placed in a 100 ml round-bottom flask, togetherwith 2.2 g of anhydrous sodium carbonate. The flask was immersed in adry ice-acetonitrile bath (at −40° C.) and 50 ml of cold (−20° C)ethanol was added. Then the mixture was stirred and allowed to stabilizeat −40° C. under an atmosphere of nitrogen. To the cold slurry wasadded, via a syringe, 9.45 g (0.0422 mol) of 2-bromoethoxysulfonylchloride over a period of 10 min. After stirring for 2 hr, the reactionmixture was allowed to warm to 15° C. and stirred for an additional 2hr. The reaction mixture was poured into 250 ml of ice-water andextracted with dichloromethane. The organic layer was washed withaqueous sodium bisulfite solution, dried over sodium sulfate andfiltered through a layer of magnesium sulfate, whereupon the solvent wasremoved on a rotary evaporator. The crude product was chromatographed ona silica gel column using 1:1 cyclohexane:ethyl acetate as the eluant togive 2.7 g (36%) of a pale yellow oil: NMR δ 2.11 (m, 3H), 2.35 (m, 1H),3.55 (m, 4H), 3.68 (m, 1H), 3.86 (m, 1H), 4.46 (m, 4H), 4.59 (m, 1H); UV(H²O) λ_(max) (ε) 252 nm (6.6 mM⁻¹ cm⁻¹).

EXAMPLE 24

This example describes the preparation ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)]ester.

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (1.03 g; 0.0068 mol) was addedto a solution of 1.33 g (0.0034 mol) of O²-(2-bromoethyl)1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate 2-bromoethylester in 35 ml of tetrahydrofuran and the resulting solution was stirredat room temperature under nitrogen. Two equivalents of thiolacetic acid(0.479 ml, 0.0068 mol) were added and the mixture was stirred at roomtemperature for 2 hr. The mixture was filtered and the solid residue waswashed with ether. The filtrate was evaporated to dryness under reducedpressure and the residue was extracted with methylene chloride. Theorganic solution was subsequently washed with ice-cold 5 N HCl, sodiumbicarbonate solution and water. The solution was dried over sodiumsulfate, filtered through a layer of magnesium sulfate and evaporated invacuo to give 710 mg of a yellow oil. Chromatography was carried out ona silica gel column eluted with 1:1 cyclohexane:ethyl acetate: UV (H₂O)λ_(max) (ε) 232 nm (7.0 mM⁻¹ cm⁻¹); NMR δ 2.09 (m, 3H), 2.36 (m, 1H),2.38 (s, 6H), 3.09 (m, 4H), 3.78 (m, 2H), 4.27 (m, 4H), 4.55 (m, 1H).

EXAMPLE 25

This example describes the determination of the halflife of the compoundproduced in Example 24 in the absence and presence of porcine liveresterase at 25° C. and pH 7.4.

A 0.009 M ethanolic stock solution ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercapto-ethyl)]ester was prepared. The decay of thiscompound was monitored at 25° C. as 1.5×10⁻⁴ M solutions in a 4 mlquartz cuvette containing 3 ml of phosphate buffer (pH 7.4) and 50 ml ofstock solution. The decay of the 232 nm chromophore was monitored on theultraviolet spectrophotometer. The halflife was estimated as 3.2 hr.

A second set of experiments was carried out using the above parametersto measure the decay after addition of 5 ml of porcine liver esterasesuspension. The half-life for the esterase reaction was 8 min at 25° C.

EXAMPLE 26

This example describes the preparation of a nitric oxide-releasingpolymer-blend ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)]ester.

A solution of 50 mg (0.132 mmol) ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)]ester in 1 ml of tetrahydrofuran wasdissolved in a solution of 498 mg of polyurethane in 10 ml oftetrahydrofuran. The homogeneous lacquer was concentrated under a streamof dry nitrogen followed by further drying under high vacuum to give asolid, which contained 0.091 mg (0.24 mmol) ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)]ester per mg of polymer composite. Rates ofNO release were measured as a function of time after immersing a 32 mgaliquot of the diazeniumdiolate in 2 ml of phosphate buffer, pH 7.4, at37° C., with a chemiluminescence detector. A set of experiments wascarried out in plain buffer, while another set was done in the presenceof porcine liver esterase. A very small amount of NO was released in theabsence of enzyme over a 200 hr period, while a significant rate of NOproduction was observed when the enzyme was present in the buffer. Thisindicates that as the diazeniumdiolate oozes out of the polymercomposite, it is hydrolyzed by the enzyme with further cleavage to NO.

EXAMPLE 27

This example describes the introduction of the nitric oxide-releasingO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)]ester into β-cyclodextrin.

β-Cyclodextrin (228 mg, 0.201 mmol) was mixed with 2 ml of water andheated to 65° C. to give a homogeneous solution. To the warm solutionwas added 76 mg (0.201 mmol) ofO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)]ester. Upon mixing, a white precipitateformed. The mixture was allowed to cool to room temperature and theproduct was collected by filtration, washed with water, and dried undervacuum to give 170 mg of product. An aqueous solution containing 33 mgof theO²-[S-acetyl-(2-mercaptoethyl)]1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate[S-acetyl-(2-mercaptoethyl)]ester: β-cyclodextrin mixture exhibited anabsorbance maximum at 232 nm and a molar absorptivity (E) of 10.8mM⁻¹cm⁻¹. Rates of NO release were measured as a function of time afterimmersing a 13 mg aliquot of the encapsulated material in 4 ml ofphosphate buffer, pH 7.4, at 37° C., with a chemiluminescence detector.A set of experiments was carried out in plain buffer, while another setwas done in the presence of porcine liver esterase. A very small amountof NO was released in the absence of enzyme over a 400 hr period while asignificant rate of NO production was observed when the enzyme waspresent in the buffer.

EXAMPLE 28

This example describes a general procedure for the preparation ofO²-glycosylated diazeniumdiolates.2,3,4,6-Tetraacetyl-α-D-glucopyranosyl bromide (acetobromoglucose) wasprepared as described in Redemann et al., Org. Syn. Coll. Vol. III:11-14 (1955). 2,3,4,6-Tetraacetyl-α-D-mannopyranosyl bromide(acetobromomannose) was prepared as described in Levene et al., J. Biol.Chem. 90: 247-250 (1931). Then, a slurry of 1 eq of a diazeniumdiolatein dimethylsulfoxide (DMSO) (0.5 mmol solid/1 ml of DMSO) was stirredwith 0.03 eq of silver oxide at room temperature under nitrogen. A 0.5 Msolution of 1.2 eq of acetobromomannose or acetobromoglucose in DMSO wasinjected dropwise and the mixture was stirred for three days. Theresulting homogeneous solution was poured into 100 ml of ice-water andextracted with ether. The ether layer was washed with water, dried oversodium sulfate and treated with charcoal. The solution was filteredthrough magnesium sulfate, concentrated on a rotary evaporator, anddried under vacuum. The glucose derivatives were purified byrecrystallization, while the glassy mannose adducts required columnchromatography.

EXAMPLE 29

This example describes the preparation of sodium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (“DEA/NO”).

A solution of 119 g (1.63 mol) of diethylamine in 100 ml of 1:1ether:acetonitrile was placed in a 500 ml Parr bottle. The solution wasdegassed, charged with 40 psi of nitric oxide, and allowed to stand atroom temperature overnight. The pressure was released and thecrystalline product was collected by filtration and dried under nitrogento give 13 g of diethylammonium1-(N,N-diethylamino)diazen-1-ium-1,2-diolate. The salt was treated with10 ml of 10 M sodium hydroxide solution and the resulting paste wastreated with 200 ml of ether to give the sodium salt. The sodium salt(“DEA/NO”) was collected by vacuum filtration, washed with ether, anddried under vacuum to give 7.1 g of product: UV (in 0.01 N NaOH) λ_(max)(ε) 250 (6.88 mM⁻¹cm⁻¹); NMR (D₂O) δ 0.96 (t, 3 H), 2.94 (q, 2 H); inDMSO-d₆ δ 0.84 (t, 3 H) and 2.75 (q, 2 H).

EXAMPLE 30

This example describes the preparation ofO²-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate.

DEA/NO (2.98 g; 0.019 mol) in DMSO was reacted with acetobromoglucose(6.9 g; 0.017 mol) as described in the general procedure of Example 28.The product was recrystallized from petroleum ether to give 5.7 g (72%)108 mg of a crystalline solid: mp 107-108° C.; UV λ_(max) (ε) 228 nm(6.92 mM⁻¹cm⁻¹); NMR δ 1.11 (t, 6 H, J=7.11), 2.02 (s, 3 H), 2.03 (s, 3H), 2.04 (s, 3 H), 2.07 (s, 3 H), 3.21 (q, 4 H, J=7.12), 3.81 (m, 1 H),4.20 (m, 2 H), 5.14 (m, 1 H), 5.33 (m, 3 H). Anal. Calcd forC₁₈H₂₉N₃O₁₁: C, 46.65; H, 6.31; N, 9.07. Found: C, 46.73; H, 6.26; N,9.01.

EXAMPLE 31

This example describes the deacylation ofO²-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)1-(N,N-diethylamino)diazen-1-ium-1,2-diolate(from Example 30).

A solution of 253 mg (0.55 mmol) of the above compound in 5 ml ofmethanol was stirred with 10 μl of 25% methanolic sodium methoxide. Theprogress of the reaction was monitored by TLC using 5:1 CH₂Cl₂:ethylacetate. The reaction was complete within 1 h at 25° C.

Dowex-50W-H⁺ resin (1 g) was added to the stirring methanolic solution.The mixture was filtered to remove the resin, and the methanolicsolution was evaporated under vacuum to give 122 mg (75%) ofO²-glucopyranosyl 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate: UVλ_(max) (ε) 228 nm (6.4 mM⁻¹ cm⁻¹); NMR (CDCl₃) δ 1.08 (t,6H), 3.23(9,4H), 5.59 (m,4H), 3.88 (m,2H), 5.29 (m,1H).

Surprisingly, the deacetylated product cleaved to the1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) anion, then to NO,only extremely slowly at pH 3, despite its acetal-like structure. Evenmore surprisingly, the cleavage proceeded extremely rapidly at pH 13.

EXAMPLE 32

This example describes the preparation of sodium1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolate.

A solution of 20 g (0.126 mol) of carboethoxy piperazine in 60 ml ofmethanol was placed in a Parr bottle. The solution was treated with 27.4ml (0.126 mol) of 25% sodium methoxide in methanol. The system wasevacuated, charged with 40 psi of nitric oxide and kept at 25° C. for 48hr. The white crystalline product was collected by filtration and washedwith cold methanol as well as with copious amounts of ether. The productwas dried under vacuum to give 14.5 g (48% yield) of sodium1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolate: mp:184-185° C.; UV (0.01 N NaOH) λ_(max) (ε) 252 nm (10 mM⁻¹ cm⁻¹); NMR(D²O) δ 1.25 (t, 3 H), 3.11 (m, 2 H), 3.68(m, 2 H), 2.15 (q, 2 H). Analcalcd. for C₆H₁₃N₄O₄Na: C 35.00%, H 5.42%, N 23.33%, Na 9.58%. Found: C34.87%, H 5.53%, N 23.26%, Na 9.69%. The half-life of this compound atpH 7 and 25° C. was estimated as 5 min. This measurement was based onthe loss of the 252 nm chromophore in the ultraviolet spectrum.

EXAMPLE 33

This example describes the preparation of O²-(glucopyranos-2-yl)1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolatetetraacetate ester.

Acetobromoglucose (2.055 g; 0.005 mol) and 1.11 g (0.00466 mol) ofsodium 1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolate werereacted as described above to give 624 mg (25%) ofQ²-(glucopyranos-2-yl)1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolatetetraacetate ester: UV λ_(max) (ε) 228 nm (7.20 mM⁻¹ cm⁻¹); NMR δ 1.26(t, 3 H), 2.02 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3 H), 2.09 (s, 3 H), 3.46(m, 4H), 3.68 (m, 4 H), 3.82 (m, 1 H), 4.17 (q, 2 H), 4.25 (m, 3 H),5.27 (m, 3 H).

EXAMPLE 34

This example describes the preparation of O²-(mannopyranos-2-yl)1-[(1-ethoxycarbonyl)piperazin-4-yl)]diazen-1-ium-1,2-diolatetetraacetate.

Acetobromomannose (10.2 g; 0.025 mol) and 5.28 g (0.022 mol) of sodium1-[(1-ethoxycarbonyl)piperazin-4-yl]diazen-1-ium-1,2-diolate werereacted as described above to give 6.4 g (53%) of a glass: UV λ_(max)(ε) 238 nm (7.5 mM⁻¹ cm⁻¹) NMR δ 1.29 (t, 3 H), 2.01 (s, 3H), 2.05 (s,3H), 2.11 (s, 3 H), 2.17 (s, 3 H), 3.13 (m, 1 H), 3.50 (m, 4 H), 3.78(m, 5 H), 4.19 (q, 2 H), 4.27(m, 3 H), 5.28 (m, 3 H), 5.42 (m, 1H).

EXAMPLE 35

This example describes the preparation of an O²-glycosylateddiazeniumdiolate directed to a mannose-fucose receptor.

Bis-[2-(N-ethoxycarbonylamino)ethyl]amine: A three-neck flask equippedwith two dropping funnels was immersed in an ice-water bath.DiethyLenetriamine (10.7 g, 0.104 mol) was placed in the cold flask anddissolved in 100 ml of 95% ethanol. To the cold solution was added 10 ml(0.205 mol) of ethylchloroformate, dropwise. A solution of 10.6 g (0.1mol) of sodium carbonate in 100 ml of distilled water was addedsimultaneously with 10 ml (0.205 mol) of ethylchloroformate. Thereaction mixture was allowed to stir at room temperature overnight. Theethanol was removed on a rotary evaporator and the aqueous portion wasextracted with dichloromethane. The organic layer was washed with water,then extracted with 5% hydrochloric acid. The organic layer containingthe neutral products was separated and set aside. The aqueous layer waswashed with dichloromethane and made basic with sodium hydroxide. Theproduct was extracted into dichloromethane, dried over sodium sulfate,filtered through magnesium sulfate and evaporated to give 4 g of acolorless oil: NMR (CDCl₃) δ 1.25 (t, 6H), 2.78 (m, 4H), 3.36 (m, 4H),4.14 (q, 4H), 5,13 (b, 2H).

Sodium1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolate: Asolution of 2.6 g (0.011 mol) ofbis-[2-(N-ethoxycarbonylamino)ethyl]amine in 20 ml of ether and 5 ml ofmethanol was placed in a 50 ml Parr bottle, treated with 2.4 ml (0.011mol) of 25% methanolic sodium methoxide, degassed, cooled to −80° C. andcharged with 50 psi of nitric oxide. A thick precipitate was observedafter 3 hr of stirring. The mixture was exposed to NO for 24 hr, thepressure was released, and the product was collected by filtration. Thesolid was washed with ether and dried under vacuum to give 1.26 g (35%)of the diazeniumdiolate: mp 170-2° C.; UV λ_(max) (ε) 252 nm (7.6 mM⁻¹cm⁻¹); NMR δ 1.24 (t, 6H), 3.19 (m, 8H), 4.11 (q, 4H).

O²-(Mannos-2-yl)1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolatetetraacetate: A partial solution of 251 mg (0.763 mmol) of sodium1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolate in2 ml of dimethylsulfoxide (DMSO) was cooled to 0° C. under nitrogen. Tothis was added 10 mg (0.06 mmol) of silver acetate, followed by the slowaddition of 1 ml of a 0.82 M solution of acetobromomannose intetrahydrofuran. The reaction mixture was allowed to stir at roomtemperature for 48 hr, poured over ice-water, and extracted with ether.The ether solution was dried over sodium sulfate, filtered through alayer of magnesium sulfate, and evaporated under vacuum to give 307 mgof an oil: UV λ_(max) 240 nm.

O²-(Mannos-2-yl)l-[bis(2-aminoethyl)amino]diazen-1-ium-1,2-diolate]:

A solution of 145 mg (0.23 mmol) of O²-(mannos-2-yl)1-[bis-{2-(N-ethoxycarbonylamino)ethyl}amino]diazen-1-ium-1,2-diolatetetraacetate in a mixture of 0.2 ml of 10 N NaOH, 2 ml of ethanol and 2ml of water was heated at reflux for 15 hr. The solution wasconcentrated under vacuum and the remaining aqueous solution wasextracted with dichloromethane. The aqueous solution was evaporated todryness under vacuum. The residue was taken up in methanol, put througha 10 g, 60 cc prepacked C-18 column, and eluted with methanol. Thefractions exhibiting an absorption maximum at 236 nm were combined andevaporated to give 32 mg of a white powder: NMR (CD₃OD) δ 2.74 (t, 4H),3.02 (t, 4H), 3.74 (m, 4H), 4.2 (m, 3H); UV λ_(max) 238 nm.

EXAMPLE 36

This example describes the preparation of a combinatorial library usingdisodium 1-(2-carboxylato)pyrrolidin-1-yl diazen-1-ium-1,2-diolate(PROLI/NO) as starting material.

The piperazine trityl resin 1, available from Calbiochem-NovabichemInt'l. (San Diego, Calif.), is treated with sulfuryl chloride to formthe chlorosulfonamide 2. Reaction of this resin with PROLI/NO givescompound 3. The free carboxylic acid can be activated to 4 by reactionwith dicyclohexyl carbodiimide (DCC) and N-hydroxysuccinimide.Nucleophilic addition of R³⁰XH (X═O, N, S) to the resin-bounddiazeniumdiolate provides a potentially large library of compounds, 5,substituted at the carboxylato portion of the molecule. Base hydrolysisof 5 frees the anionic diazeniumdiolate 6 from the resin. This library,6, may now be reacted with electrophiles R³¹X to form new sets ofcompound having structure 7.

All publications, patents and patent applications, cited herein arehereby incorporated by reference to the same extent as if eachpublication were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

What is claimed is:
 1. An O²-substituted diazeniumdiolate having theformula

wherein X is selected from the group consisting of an amino, apolyamino, a C₁-C₂₄ aliphatic, a C₆-C₃₀ aryl, a C₃-C₃₀ heteroaromatichaving 1 to 3 heteroatorms selected from the group consisting of oxygen,nitrogen and sulfur, a C₃-C₃₀ nonaromatic cyclic, and an oximyl, and Qis selected from the group consisting of an acridinyl, an anthracenyl, abenzimidazolyl, a benzisoxazolyl, a benzofuryl, a benzothienyl, abenzoxazolyl, a benzopyrazolyl, a benzothiazolyl, a carbazolyl, achlorophyllyl, a cinnolyl, a furyl, an imidazolyl, an indolyl, anisobenzofuryl, an isoindolyl, an isoxazolyl, an isothiazolyl, anisoquinolyl, a naphthyl, an oxazolyl, a phenanthryl, a phenanthridinyl,a phenothiazinyl, a phenoxazinyl, a phenyl, a phthalimidyl, aphthalazinyl, a phthalocyaninyl, a pteridinyl, a purinyl, whichisoptionally part of a nucleic acid, a ribosylpurinyl, a pyrazinyl, apyrazolyl, a pyridazyl, a pyridyl, a pyrimidyl, which is optionally partof a nucleic acid, a ribosylpyrimidyl, a pyrrocolinyl, a pyrryl, aquinolyl, a quinoxalinyl, a quinazolinyl, a sydnonyl, a tetrazolyl, athiazolyl, a thienyl, a thyroxinyl, a triazinyl, and a triazolyl,wherein a ring atom of Q is bonded to the O²-oxygen, wherein X and Q areoptionally substituted, with the proviso that, when Q is an imidazolyl,X is not an imidazolyl.
 2. The diazeniumdiolate of claim 1, wherein Q ispart of a vitamin.
 3. The diazeniumdiolate of claim 1, wherein Q is partof a hormone.
 4. The diazeniumdiolate of claim 1, wherein Q is apyrimidyl, which, optionally, is part of a nucleic acid.
 5. Thediazeniumdiolate of claim 4, wherein Q is a ribosylpyrimidyl.
 6. Thediazeniumdiolate of claim 1, wherein Q is a purinyl, which, optionally,is part of a nucleic acid.
 7. The compound of claim 6, wherein Q is aribosylpurinyl.
 8. The diazeniumdiolate of claim 1, wherein X is linkedto the N¹ nitrogen through an atom other than a carbon atom.
 9. Thediazeniumdiolate of claim 1, wherein X is substituted with one or moremoieties selected from the group consisting of —[N(NO)O⁻], a halo,hydroxy, an alkylthio, an alkoxy, an aryloxy, an amino, cyano, asulfonato, mercapto, nitro, a C₁-C₁₂ aliphatic, a C₃-C₈ cycloalkyl, aC₃-C₈ heterocyclic, a C₂-C₁₂ olefinic, benzyl, phenyl, benzylcarbonyl,phenylcarbonyl, glucosyl, ribosyl, glucosyl, mannosyl, deoxyribosyl,dextranyl, starch, glycogenyl, lactosyl, fucosyl, galactosyl, fructosyl,glucosaminyl, galactosaminyl, heparinyl, maltosyl, sucrosyl, sialyl,cellulose, phosphorylated pentosyl, polyphosphorylated pentosyl,phosphorylated hexosyl, polyphosphorylated hexosyl, phosphono,phosphato, and phosphato in which one or more oxygen atoms areindependently replaced with S or NR¹, wherein R¹ is a C₁-C₈ aliphatic, aC₃-C₈ cycloalkyl, a C₆-C₈ aryl, or a C₃-C₈ heteroaromatic having 1 to 3heteroatoms selected from the group consisting of oxygen, nitrogen andsulfur.
 10. The diazeniumdiolate of claim 1, wherein Q is substitutedwith one or more moieties selected from the group consisting ofX[N(O)NO]⁻, wherein X is as defined in claim 1, halo, hydroxy,alkylthio, arylthio, alkoxy, aryloxy, amino, mono- or di-substitutedamino, ammonio, substituted ammonio, nitroso, cyano, sulfonato,mercapto, nitro, oxo, a C₁-C₂₄ aliphatic, a C₂-C₁₂ olefinic, a C₃-C₂₄cycloalkyl, a C₃-C₂₄ heterocyclic, benzyl, phenyl, substituted benzyl,substituted phenyl, benzylcarbonyl, phenylcarbonyl, glucosyl, ribosyl,glucosyl, mannosyl, deoxyribosyl, dextranyl, starch, glycogenyl,lactosyl, fucosyl, galactosyl, fructosyl, glucosaminyl, galactosaminyl,heparinyl, maltosyl, sucrosyl, sialyl, cellulose, phosphorylatedpentosyl, polyphosphorylated pentosyl, phosphorylated hexosyl,polyphosphorylated hexosyl, substituted benzylcarbonyl, substitutedphenylcarbonyl, phosphono, phosphato, and phosphato in which one or moreoxygen atoms are independently replaced with S or NR¹, wherein R¹ is aC₁-C₁₀ aliphatic, a C₃-C₁₀ cycloalkyl, a C₆-C₁₀ aryl, or a C₁-C₁₀heteroaromatic having 1 to 3 heteroatoms selected from the groupconsisting of oxygen, nitrogen and sulfur.
 11. The diazeniumdiolate ofclaim 1, having the formula

wherein b and d can be the same or different and may be zero or one, R¹,R², R³, R⁴, and R⁵ are the same or different and are selected from thegroup consisting of hydrogen, C₃-C₈ cycloalkyl, C₁₋₁₂ straight orbranched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl,trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or2,2,2-trihalo-t-butoxycarbonyl, and i, j, and k are the same ordifferent and are integers from 2 to
 12. 12. The diazeniumdiolate ofclaim 1, having the formula

wherein D is

and wherein R¹⁰ and R¹¹ are the same or different and are selected fromthe group consisting of hydrogen, C₃₋₈ cycloalkyl, C₁₋₁₂ straight orbranched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl,trifluoroacetyl, p-toluyl, t-butoxycarbonyl, and2,2,2-trichloro-t-butoxycarbonyl, and f is an integer from 0 to
 12. 13.The diazeniumdiolate of claim 1, having the formula:

wherein R⁶ and R⁷ can be the same or different and are H, a C₁-C₁₂straight chain alkyl, a C₁-C₁₂ alkoxy or acyloxy substituted straightchain alkyl, a C₂-C₁₂ hydroxy or halo substituted straight chain alkyl,a C₃-C₁₂ branched chain alkyl, a C₃-C₁₂ hydroxy, halo, alkoxy, oracyloxy substituted branched chain alkyl, a C₂-C₁₂ straight chainolefinic, or a C₃-C₁₂ branched chain olefinic, wherein R⁶ and R⁷ areoptionally substituted with hydroxy, alkoxy, acyloxy, halo or benzyl; orR⁶ and R⁷ together with the nitrogen atom to which they are bonded forma heterocyclic ring selected from the group consisting of:

wherein A is N, O, or S, w is 1 to 12, y is 1 or 2, z is 1 to 5, R⁸ ishydrogen, a C₁-C₈ straight chain alkyl, a C₃-C₈ branched chain alkyl, aC₃-C₈ cycloalkyl, a C₆-C₃₀ aryl, a C₃-C₃₀ heteroaromatic having 1 to 3heteroatoms selected from the group consisting of oxygen, nitrogen andsulfur, and R⁹ is hydrogen, a C₁-C₆ straight chain alkyl or a C₃-C₆branched chain alkyl.
 14. The diazeniumdiolate of claim 13, wherein R⁶is hydrogen.
 15. The diazeniumdiolate of claim 13, wherein R⁶ and R⁷ areethyl and Q is selected from the group consisting of:


16. An O²-substituted diazeniumdiolate having the formula

wherein X is an inorganic moiety that results in a compound of Formula(I) capable of disassociating under physiological conditions and Q isselected from the group consisting of an acridinyl, an anthracenyl, abenzimidazolyl, a benzisoxazolyl, a benzofuryl, a benzothienyl, abenzoxazolyl, a benzopyrazolyl, a benzothiazolyl, a carbazolyl, achlorophyllyl, a cinnolyl, a furyl, an imidazolyl, an indolyl, anisobenzofuryl, an isoindolyl, an isoxazolyl, an isothiazolyl, anisoquinolyl, a naphthyl, an oxazolyl, a phenanthryl, a phenanthridinyl,a phenothiazinyl, a phenoxazinyl, a phenyl, a phthalimidyl, aphthalazinyl, a phthalocyaninyl, a pteridinyl, a purinyl, which isoptionally part of a nucleic acid, a ribosylpurinyl, a pyrazinyl, apyrazolyl, a pyridazyl, a pyridyl, a pyrimidyl, which is optionally partof a nucleic acid, a ribosylpyrimidyl, a pyrrocolinyl, a pyrryl, aquinolyl, a quinoxalinyl, a quinazolinyl, a sydnonyl, a tetrazolyl, athiazolyl, a thienyl, a thyroxinyl, a triazinyl, and a triazolyl;wherein a ring atom of Q is bonded to the O²-oxygen, wherein X and Q areoptionally substituted.
 17. The diazeniumdiolate of claim 16, wherein Xis ⁻O— or ⁻O₃S—.
 18. A composition comprising a diazeniumdiolate ofclaim 1 and a carrier.
 19. The composition of claim 18, wherein the ringatom of Q bonded to said O²-oxygen is carbon or nitrogen.
 20. Acomposition comprising a diazeniumdiolate of claim 16, and a carrier.21. The composition of claim 20, wherein the ring atom of Q bonded tosaid O²-oxygen is carbon or nitrogen.
 22. A method of treating orpreventing a biological disorder in an animal, wherein said disorder isselected from the group consisting of angina, acute myocardialinfarction, congestive heart failure, hypertension and metastasis, whichmethod comprises administering to said animal an amount of a compound ofclaim 1 or a diazeniumdiolate of formula

wherein X and Q are imidazoles, sufficient to treat or prevent thebiological disorder in said animal.
 23. The method of claim 22, whereinsaid biological disorder is due to hypertension.
 24. The method of claim22, wherein said biological disorder is due to acute myocardialinfarction.
 25. The method of claim 22, wherein said biological disorderis due to metastasis.
 26. A method of treating or preventing abiological disorder in an animal, wherein said disorder is selected fromthe group consisting of angina, acute myocardial infarction, congestiveheart failure, hypertension and metastasis, which method comprisesadministering to said animal an amount of a compound of claim 16sufficient to treat or prevent the biological disorder in said animal.27. A method of treating or preventing a biological disorder in amammal, wherein said disorder is selected from the group consisting ofangina, acute myocardial infarction, congestive heart failure,hypertension and metastasis, which method comprises administering to theanimal a compound of claim 1 or a diazeniumdiolate of formula

in which X and R are compounds comprising a pyranose ring or a furanosering, wherein the compounds are attached to the O² of thediazeniumdiolate by the 2 position of the pyranose ring or the furanosering, in an amount sufficient to treat or prevent the biologicaldisorder.
 28. The diazeniumdiolate of claim 16, wherein Q is part of avitamin.
 29. The diazeniumdiolate of claim 16, wherein Q is part of ahormone.
 30. A method of treating an animal infected with an infectiousagent comprising a zinc finger protein that can be inactivated by nitricoxide, the method comprising administering to said animal an amount of acompound of claim 1, 16 or a diazeniumdiolate of formula

wherein X and Q are imidazoles, sufficient to inactivate the zinc fingerprotein in the infectious agent so as to treat the infection in theanimal.
 31. A method of treating an animal for cancer, wherein thecancer involves a zinc finger protein that can be inactivated by nitricoxide, the method comprising administering to the animal an amount of acompound of claim 1, 16 or a diazeniumdiolate of formula

wherein X and Q are imidazoles, sufficient to inactivate the zinc fingerprotein so as to treat the cancer in said animal.
 32. A method oftreating an animal for cancer, wherein the cancer is resistant totreatment with a chemotherapeutic agent, the method comprisingadministering to the animal an amount of a compound of claim 1, 16 or adiazeniumdiolate of formula

wherein X and Q are imidazoles, sufficient to render the cancer in theanimal treatable with the chemotherapeutic agent.
 33. A method ofmodulating steroid hormone activity in a mammal, wherein the mammal isin need of modulation of steroid hormone and wherein the animal has asteroid hormone receptor comprising a zinc finger protein which can beinactivated by nitric oxide, the method comprising administering to theanimal an amount of a compound of claim 1, 16 or a diazeniumdiolate offormula.

wherein X and Q are imidazoles, sufficient to inactivate the steroidhormone receptor protein so as to modulate steroid hormone activity inthe mammal.
 34. The method of claim 30, wherein the infectious agent isa virus.
 35. The method of claim 34, wherein the virus is HIV.
 36. Themethod of claim 30, wherein said infectious agent is a parasite.
 37. Themethod of claim 36, wherein said parasite is Giardia.
 38. The method ofclaim 32, wherein the chemotherapeutic agent is a DNA-damaging agent.39. The method of claim 38, wherein the DNA-damaging agent is selectedfrom the group consisting of an alkylating agent and an oxidizing agent.